Human papilloma virus immunoreactive peptides

ABSTRACT

This invention provides immunogenic peptides from the HPV-18E6 protein that comprise class I restricted T cell epitopes and discloses methods of administering these peptides to individuals, and a method for monitoring or evaluating an immune response to HPV with these peptides.

CROSS-REFERENCES TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. provisional patentapplication Serial No. 60,278,520, filed Mar. 23, 2001. The 60/278,520provisional patent application is herein incorporated by its referencein its entirety.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDERFEDERALLY SPONSOREDRESEARCH AND DEVELOPMENT

[0002] This invention is owned by the United States Government.

FIELD OF THE INVENTION

[0003] This invention relates to treatment of human papilloma virus(HPV) infection and in particular it relates to immunogenic peptideswhich are suitable for use in vaccines. This invention also relates tomethods of using immunogenic peptides suitable for stimulating in vitro,lymphocytes or antigen presenting cells previously isolated from apatient and returning these stimulated cells to the patient. Thisinvention relates to methods of monitoring the immune response in apatient exposed to these immunogenic peptides.

BACKGROUND OF THE INVENTION

[0004] Papilloma viruses are non-enveloped DNA viruses with a doublestranded circular genome of approximately 8,000 bp. Over 75 types ofhuman papilloma viruses (HPV) have been typed at the DNA level, andthese can be broadly grouped into families on the basis of their tissuetropism.

[0005] Histologic, molecular, and epidemiologic evidence have implicatedsome HPV strains in cervical dysplasia and cervical cancer. Many studiessupport the view that most moderate and severe cervical intraepithelialneoplasias (CIN) contain HPV DNA which is exclusively detected in thehistologically abnormal epithelium of these lesions. Persistentinfection with HPV is believed to be the predominant risk factor fordevelopment of cervical carcinoma. HPV DNA is readily found in episomalform within cells exhibiting a cytopathic effect, while the HPV DNA isfound integrated within the chromosomes of cells associated with mosthigh grade pre-cancerous lesions and cancer. Approximately 23 HPV typesare commonly found in anogenital screening programs, but only 10-15 areassociated with progressive disease. Type 16 is the type most commonlyfound in cervical cancer tissue.

[0006] Papillomaviruses contain nine open reading frames. HPV genes withtransforming properties have been mapped to open reading frames E6 andE7. Substantial biochemical work has demonstrated that the HPV E6protein inactivates the protein p53, whereas the E7 protein interfereswith retinoblastoma (Rb) protein function. Since p53 and Rb aretumor-suppressor proteins which function as cell division inhibitors,their inactivation by E6 and E7 leads the cell to enter into S phase ofthe cell cycle. Expression of E6 and E7 is sufficient to immortalizesome primary cell lines, and blocking E6 or E7 function has been shownto reverse the transformed state.

[0007] Abundant circumstantial evidence implicates host immunemechanisms in the control of HPV associated tumours of the anogenitalepithelium (Singer et al., British Medical Journal 288: 735-736, 1984).There is an increased incidence of pre-neoplastic (Frazer et al., Lancetii 657-660, 1986) and neoplastic associated lesions in homosexual menimmunosuppressed by human immunodeficiency virus infection and amarkedly increased risk of squamous cell carcinoma (SCC) of the cervixand vulva but not of control organs such as breast and rectum inimmunosuppressed allograft recipients (Sheil and Plavel Ninth Report ofAustralian and New Zealand Combined Dialysis and Transplant Registry pp104-112 Edited by APS Disney 1986).

[0008] Taken with the above, the normal natural history of HPV infectionin most patients with alpha-gamma globulinemia suggests that cellularrather than humoral responses are important for the control of thephenotypic expression of HPV infection (Kirschner Progress in MedicalVirology, 1986).

[0009] Standard immunological approaches to the study of anogenital HPVinfection have been hampered by the lack of a suitable animal model andof an in vitro epithelial cell culture permissive for HPV. Vaccines havealso been proposed in regard to HPV with however only indifferentsuccess.

[0010] It has been proposed to use vaccines containing autogenous tumorhomogenates (Abcarian et al., J. Surg Res 22: 231-236, 1977, Dis. ColonRectum 25:648-51, 1982, Dis. Colon Rectunt 19: 237-244, 1976. However ithas recently been advocated that patients should no longer be treatedwith autogenous vaccines because of the potential oncogenic effect ofthe viral DNA (Bunney Br Med. J293:1045-1047, 1986).

[0011] Data on successful prophylactic vaccination exist only for bovinefibropapillomas homogenized homogenate of bovine fibropapillomas and hasbeen shown to provide limited immunity (Olson et al., J Am Vet Med.Assoc. 135: 499, 1959, Cancer Res 22: 463, 1962). A vaccine including anengineered L1 fusion protein (Pilacinski et al., UCLA Symp. Molecularand Cellular Biology New Series Vol 32 Papilloma Viruses Molecular andClinical Aspects Alan R Liss N.Y., pg. 257,1985) has also been used incalves but proved unsuccessful in humans. In Pfister, PAPILLOMA VIRUSESAND HUMAN CANCER, CRC Press Inc. (1990) it is stated that there ispresently no evidence for a possible prevention of HPV infection by theuse of a capsid protein vaccine, but induction of an anti-tumor cellimmunity appears to be feasible.

[0012] The L1 and L2 genes have been the basis of vaccines for theprevention and treatment of papilloma virus infections and immunogensused in the diagnosis and detection of papilloma viruses (InternationalPatent Specifications WO 86/05816 and E 08303623). However, it appearsthat no commercial usage of these vaccines have taken place.

[0013] Reference may also be made to Patent Specification EP 386734which describes new immunogenic regions of HPV-16 E7 protein which maybe useful in vaccines, EP 375555 which describes HPV-16 peptides usefulas immunoassay reagents for the detection of HPV-16 proteins and whichcontain an antigenic determinant for HPV16, a reference in VACCINE 83:199-204 (1990) which describes vaccines including recombinantsexpressing HPV E5, E6 or E7 ORF intended for use in providing antitumoractivity, Australian Specification 52860/90 which describes screeningantibodies for specificity to an antigen which is an epitope of HPV-16L1 or E7 proteins, Australian Specification 75535/87 which describessynthetic peptides of HPV corresponding to an amino acid sequence regionhaving at least one reverse turn and predicted hydrophilicity, PatentSpecification EP 217919 which describes type specific papillomavirus DNAsequences and peptides useful in vaccines containing 15-75 nucleotides,U.S. Pat. No. 4,551,270 which describes at least one antigenicdeterminant of papillomavirus and immunogens and vaccines containing theantigenic determinant, Patent Specification EP 412762 which describes apolypeptide, which inhibits binding of the HPV E7 protein toretinoblastoma gene which may be used in vaccines for treatment ofcervical cancer and genital warts, French Specification 2643817 whichdescribes a vaccine for treatment of tumours induced by papillomaviruscontaining recombinant poxvirus with heterologous DNA encoding region ofnon structural papillomavirus, Japanese Specification J01061665 whichdescribes antibodies formed to an antigen polypeptide of HPV-16E6 or E7protein, Australian Specification 76018/87 which describes expressionproducts of HPV-16 or HPV-18 which may be used for the production ofantibodies, EP235187 which describes kits containing polypeptide(s)expressed by several groups of papilloma virus including HPV-16 andHPV-18 which are expression products of E6, E7 or L2 genes and U.S. Pat.No. 4,777,239 which includes, diagnostic synthetic peptides for HPV oneof which includes residues 45-58 of protein E6 and 40-50 of protein E7which may be used as a therapeutic agent.

[0014] Virus-specific, human leukocyte antigen (HLA) class I-restrictedcytotoxic T lymphocytes (CTL) are known to play a major role in theprevention and clearance of virus infections in vivo (Oldstone et al,Nature 321:239, 1989; Jamieson et al., J. Virol. 61:3930, 1987; Yap etal, Nature 273:238, 1978; Lukacher et al., J. Exp. Med. 160:814, 1994;McMichael et al., N. Engl. J. Med. 309:13, 1983; Sethi et al., J. Gen.Virol. 64:443, 1983; Watari et al., J. Exp. Med. 165:459, 1987; Yasukawaet al, J. Immunol. 143:2051, 1989; Tigges et al., J. Virol. 66:1622,1993; Reddenhase et al. J. Virol. 55:263, 1985; Quinnan et al., N. Engl.J. Med. 307:6, 1982). HLA class I molecules are expressed on the surfaceof almost all nucleated cells. Following intracellular processing ofantigens, epitopes from the antigens are presented as a complex with theHLA class I molecules on the surface of such cells. CTL recognize thepeptide-HLA class I complex, which then results in the destruction ofthe cell bearing the HLA-peptide complex directly by the CTL and/or viathe activation of non-destructive mechanisms e.g., the production ofinterferon, that inhibit viral replication.

SUMMARY OF TIIE INVENTION

[0015] This invention applies our knowledge of the mechanisms by whichantigen is recognized by T cells, for example, to develop epitope-basedvaccines directed towards HPV. More specifically, this applicationcommunicates our discovery of specific epitope pharmaceuticalcompositions and methods of use in the prevention and treatment of HPVinfection.

[0016] Upon development of appropriate technology, the use ofepitope-based vaccines has several advantages over current vaccines,particularly when compared to the use of whole antigens in vaccinecompositions. There is evidence that the immune response to wholeantigens is directed largely toward variable regions of the antigen,allowing for immune escape due to mutations. The epitopes for inclusionin an epitope-based vaccine are selected from conserved regions of viralor tumor-associated antigens, which thereby reduces the likelihood ofescape mutants. Furthermore, immunosuppressive epitopes that may bepresent in whole antigens can be avoided with the use of epitope-basedvaccines.

[0017] An additional advantage of an epitope-based vaccine approach isthe ability to combine selected epitopes (CTL and HTL), and further, tomodify the composition of the epitopes, achieving, for example, enhancedimmunogenicity. Accordingly, the immune response can be modulated, asappropriate, for the target disease. Similar engineering of the responseis not possible with traditional approaches.

[0018] Another major benefit of epitope-based immune-stimulatingvaccines is their safety. The possible pathological side effects causedby infectious agents or whole protein antigens, which might have theirown intrinsic biological activity, is eliminated.

[0019] An epitope-based vaccine also provides the ability to direct andfocus an immune response to multiple selected antigens from the samepathogen. Thus, patient-by-patient variability in the immune response toa particular pathogen may be alleviated by inclusion of epitopes frommultiple antigens from that pathogen in a vaccine composition. A“pathogen” may be an infectious agent or a tumor associated molecule.

[0020] One of the most formidable obstacles to the development ofbroadly efficacious epitope-based immunotherapeutics, however, has beenthe extreme polymorphism of HLA molecules. To date, effectivenon-genetically biased coverage of a population has been a task ofconsiderable complexity; such coverage has required that epitopes beused that are specific for HLA molecules corresponding to eachindividual HLA allele, therefore, impractically large numbers ofepitopes would have to be used in order to cover ethnically diversepopulations. Thus, there has existed a need for peptide epitopes thatare bound by multiple HLA antigen molecules for use in epitope-basedvaccines. The greater the number of HLA antigen molecules bound, thegreater the breadth of population coverage by the vaccine.

[0021] In a preferred embodiment, epitopes for inclusion in vaccinecompositions and the methods of the invention are found in Table 1.

[0022] Furthermore, as described herein in greater detail, a need hasexisted to modulate peptide binding properties, for example, so thatpeptides that are able to bind to multiple HLA antigens do so with anaffinity that will stimulate an immune response. Identification ofepitopes restricted by more than one HLA allele at an affinity thatcorrelates with immunogenicity is important to provide thoroughpopulation coverage, and to allow the elicitation of responses ofsufficient vigor to prevent or clear an infection in a diverse segmentof the population. Such a response can also target a broad array ofepitopes.

[0023] The invention also includes an embodiment comprising a method formonitoring or evaluating an immune response to HPV in a patient having aknown HLA-type, the method comprising incubating a T lymphocyte samplefrom the patient with a peptide composition comprising an HPV epitopeconsisting essentially of an amino acid sequence described in Table 1which binds the product of at least one HLA allele present in saidpatient, and detecting the presence of a T lymphocyte that binds to thepeptide.

[0024] A method of inducing a cytotoxic T lymphocyte response againsthuman papilloma virus 16 (HPV 16) in a patient is provided, the methodcomprising contacting cytotoxic T cells from a patient with animmunogenic peptide of 20 amino acid residues or less comprising across-reactive peptide from the E6 protein of a related HPV strain, HPV18, that has higher affinity for the HLA-A2.1 molecule than thecorresponding epitope from HPV 16 itself, said peptide comprising thesequence X₁KLPDLCTEL(SEQ ID NO:1) X₂, wherein X₁ and X₂ are peptides of0-11 amino acids in length comprising either native or non-native aminoacid sequence and returning said cytotoxic T cells to the patient in anamount sufficient to induce a cytotoxic T cell response. X₁ or X₂ cancomprise an HLA binding motif other than HLA-A2. X₁ or X₂ can comprisean amino acid sequence capable of binding to an HLA class II molecule.The peptide can be bound to an HLA molecule on an antigen presentingcell or the peptide can be bound to an HLA molecule on a lymphocyte. TheHLA molecule can be HLA-A2, or the HLA molecule can be an HLA moleculeother than HLA-A2.

[0025] A method for inducing an immune response against human papillomavirus 16 (HPV 16) is provided, comprising administering to a subject acomposition, which is selected from a group consisting of:

[0026] (i) a peptide of 20 amino acids or less comprising across-reactive peptide from the E6 protein of a related HPV strain, HPV18, that has higher affinity for the HLA-A2.1 molecule than thecorresponding epitope from HPV 16 itself, said peptide comprising thesequence X₁KLPDLCTEL(SEQ ID NO:1)X₂, wherein X₁ and X₂ are peptides of0-11 amino acids in length comprising either native or non-native aminoacid sequences; (ii) an antigen presenting cell pulsed with saidpeptide; and (iii) a cell sensitized in vitro to said peptide. Thecomposition can be in a pharmaceutically acceptable carrier or in asterile medium. The method can further comprise co-administering to thesubject an immune adjuvant selected from non-specific immune adjuvants,subcellular microbial products and fractions, haptens, immunogenicproteins, immunomodulators, interferons, thymic hormones and colonystimulating factors. The administration step can comprise sensitizingCD8+ cells in vitro to said peptide and administering the sensitizedcells to the subject in a sterile medium.

[0027] A vaccine is provided for preventing or treating human papillomavirus 16 (HPV 16) infection that induces a protective or therapeuticimmune response, wherein said vaccine comprises a peptide of 20 aminoacids or less comprising a cross-reactive peptide from the E6 protein ofa related HPV strain, HPV 18, that has higher affinity for the HLA-A2.1molecule than the corresponding epitope from HPV 16 itself, said peptidecomprising the sequence X₁KLPDLCTEL(SEQ ID NO:1)X₂, wherein X₁ and X₂are peptides of 0-11 amino acids in length comprising either native ornon-native amino acid sequences and a pharmaceutically acceptablecarrier. The vaccine can further comprise co-administering to thesubject an immune adjuvant selected from non-specific immune adjuvants,subcellular microbial products and fractions, haptens, immunogenicproteins, immunomodulators, interferons, thymic hormones and colonystimulating factors. The peptide can be administered by administering toa subject an expression vector that expresses said peptide.

[0028] A method for monitoring or evaluating an immune response to humanpapilloma virus 16 (HPV 16) in a patient having the HLA-A2.1 type isprovided, the method comprising incubating a T lymphocyte sample fromthe patient with a peptide of 20 amino acids or less comprising across-reactive peptide from the E6 protein of a related HPV strain, HPV18, that has higher affinity for the HLA-A2.1 molecule than thecorresponding epitope from HPV 16 itself, said peptide comprising thesequence X₁KLPDLCTEL(SEQ ID NO:1)X₂, wherein X₁ and X₂ are peptides of0-11 amino acids in length comprising either native or non-native aminoacid sequences and which peptide bears a binding motif corresponding toat least one HLA allele present in said patient, and detecting thepresence of a T lymphocyte that recognizes the peptide.

[0029] A method screening for exposure to human papilloma virus 16 (HPV16) in a patient having the HLA-A2.1 type is also provide, the methodcomprising incubating a T lymphocyte sample from the patient with apeptide of 20 amino acids or less comprising a cross-reactive peptidefrom the E6 protein of a related HPV strain, HPV 18, that has higheraffinity for the HLA-A2.1 molecule than the corresponding epitope fromHPV 16 itself, said peptide comprising the sequence X₁KLPDLCTEL(SEQ IDNO:1)X₂, wherein X₁ and X₂ are peptides of 0-11 amino acids in lengthcomprising either native or non-native amino acid sequences and whichpeptide bears a binding motif corresponding to at least one HLA allelepresent in said patient, and detecting the presence of a T lymphocytethat recognizes the peptide, the presence of such a T lymphocyteindicating exposure to HPV.

[0030] A method is provided for inducing a cytotoxic T lymphocyteresponse and protective immunity against tumors induced by humanpapilloma virus 16 (HPV 16) using a cross-reactive peptide from the E6protein of a related HPV strain, HPV 18, that has higher affinity forthe HLA-A2.1 molecule than the corresponding epitope from PV 16 itself,said HPV18 peptide having the sequence X₁KLPDLCTEL(SEQ ID NO:1)X₂,wherein X₁ and X₂ are peptides of 0-11 amino acids.

[0031] As will be apparent from the discussion below, other methods andembodiments are also contemplated. Further, novel synthetic peptidesproduced by any of the methods described herein are also part of theinvention.

BRIEF DESCRIPTION OF THE DRAWINGS

[0032]FIG. 1. HPV-18E6 (13-21) peptide binding to 12 cells show highaffinity to HLA-A2 and is found to be higher than the homologousHPV-16E6 peptide.

[0033]FIG. 2. Lymphocytes obtained from HLA-A2 patients were stimulatedin vitro with HPV-18E6 peptides were found to specifically lyseautologous PBMC pulsed with either HPV-16E6 or HPV-18E6 peptides.Similarly T cells stimulated with HPV-16E6 peptides were found to lyseautologous PBMC pulsed with either HPV-16 E6 or HPV-18E6 peptides.

DETAILED DESCRIPTION OF THE INVENTION

[0034] The present invention provides an HPV-18E6 (13-21) peptide,KLPDLCTEL (SEQ ID NO:1), with a predicted HLA-A2 binding motifunderlined, and shows that this peptide has a higher affinity for theHLA-A2.1 molecule than the corresponding epitope from HPV 16 itself.

[0035] The peptide epitopes in Table 1 and corresponding nucleic acidcompositions of the present invention are useful for stimulating animmune response to HPV by stimulating the production of CTL or HTLresponses. The peptide epitopes, which are derived directly orindirectly from native HPV amino acid sequences, are able to bind to HLAmolecules and stimulate an immune response to HPV. The complete proteinsequence from HPV-18E6 can be obtained from Genbank. Peptide epitopesand analogs thereof can also be readily determined from sequenceinformation that may subsequently be discovered for heretofore unknownvariants of HPV, as will be clear from the disclosure provided below.

[0036] The peptide epitopes of the invention have been identified. Alsodiscussed below is that analog peptides have been derived and thebinding activity for HLA molecules modulated by modifying specific aminoacid residues to create peptide analogs exhibiting alteredimmunogenicity. Further, the present invention provides compositions andcombinations of compositions that enable epitope-based vaccines that arecapable of interacting with HLA molecules encoded by various geneticalleles to provide broader population coverage than prior vaccines.

[0037] Definitions

[0038] The invention can be better understood with reference to thefollowing definitions, which are listed alphabetically:

[0039] An “antigen-presenting cell” is a specialized cell that expressclass II MHC proteins on its cell surface. Short peptides associatenon-covalently with the surface class II MHC proteins which are thendetected by other T cells such as T helper cells (HTL or helper Tlymphocytes). Types of antigen presenting cells include, macrophages, Bcells, and dendritic cells.

[0040] A “cytotoxic T cell” is a cell which will kill another cell thathas foreign macromolecules on its surface. Frequently these foreignmacromolecules will be peptides non-covalently bound to cell surfaceclass I MHC molecules. Most, but not all cytotoxic T cells expresssurface CD8 protein. A small percentage of cytotoxic T cells express CD4on their cell surface and a small percentage of cytotoxic T cells do notexpress either CD4 or CD8 on their cell surface. Cytotoxic T cell, CTLand Tc cell will be used interchangeably herein.

[0041] An “epitope” is a set of amino acid residues which is involved inrecognition by a particular immunoglobulin, or in the context of Tcells, those residues necessary for recognition by T cell receptorproteins and/or Major Histocompatibility Complex (MHC) receptors. In animmune system setting, in vivo or in vitro, an epitope is the collectivefeatures of a molecule, such as primary, secondary and tertiary peptidestructure, and charge, that together form a site recognized by animmunoglobulin, T cell receptor or HLA molecule. Throughout thisdisclosure epitope and peptide are often used interchangeably.

[0042] It is to be appreciated that protein or peptide molecules thatcomprise an epitope of the invention as well as additional amino acid(s)are still within the bounds of the invention. In certain embodiments,there is a limitation on the length of a peptide of the invention whichis not otherwise a construct. An embodiment that is length-limitedoccurs when the protein/peptide comprising an epitope of the inventioncomprises a region (i.e., a contiguous series of amino acids) having100% identity with a native sequence. In order to avoid the definitionof epitope from reading, e.g. on whole natural molecules, there is alimitation on the length of any region that has 100% identity with anative peptide sequence. Thus, for a peptide comprising an epitope ofthe invention and a region with 100% identity with a native peptidesequence (and is not otherwise a construct), the region with 100%identity to a native sequence generally has a length as indicated.

[0043] “Human Leukocyte Antigen” or “HLA” is a human class I or class IIMajor Histocompatibility Complex (MHC) protein (see, e.g., Stites, etal., IMMUNOLOGY, 8^(TH) ED., Lange Publishing, Los Altos, Calif. (1994).

[0044] Peptide binding may be determined using assay systems includingthose using: live cells (e.g., Ceppellini et al., Nature 339:392, 1989;Christnick et al., Nature 352:67, 1991; Busch et al., Int. Immunol.2:443, 1990; Hill et al., J. Immunol. 147:189, 1991; del Guercio et al.,J. Immunol. 154:685, 1995), cell free systems using detergent lysates(e.g., Cerundolo et al., J. Immunol. 21:2069, 1991), immobilizedpurified MHC (e.g., Hill et al., J. Immunol. 152,2890, 1994; Marshall etal., J. Immunol. 152:4946, 1994), ELISA systems (e.g., Reay et al., EMBOJ. 11:2829, 1992), surface plasmon resonance (e.g., Khilko et al., J.Biol. Chem. 268:15425, 1993); high flux soluble phase assays (Hammer etal., J. Exp. Med. 180:2353, 1994), and measurement of class I MHCstabilization or assembly (e.g., Ljunggren et al., Nature 346:476, 1990;Schumacher et al., Cell 62:563, 1990; Townsend et al, Cell 62:285, 1990;Parker et al., J. Immunol. 149:1896, 1992, which are incorporated hereinby reference in their entireties, but also for their teaching regardingpeptide binding assays).

[0045] The terms “identical” or percent “identity,” in the context oftwo or more peptide sequences, refer to two or more sequences orsubsequences that are the same or have a specified percentage of aminoacid residues that are the same, when compared and aligned for maximumcorrespondence over a comparison window, as measured using a sequencecomparison algorithm or by manual alignment and visual inspection.

[0046] An “immunogenic peptide” or “peptide epitope” is a peptide thatcomprises an allele-specific motif or supermotif such that the peptidewill bind an HLA molecule and induce a CTL and/or HTL response. Thus,immunogenic peptides of the invention are capable of binding to anappropriate HLA molecule and thereafter inducing an HLA-restrictedcytotoxic or helper T cell response to the antigen from which theimmunogenic peptide is derived.

[0047] The phrases “isolated” or “biologically pure” refer to materialwhich is substantially or essentially free from components whichnormally accompany the material as it is found in its native state.Thus, isolated peptides in accordance with the invention preferably donot contain materials normally associated with the peptides in their insitu environment An “isolated” epitope refers to an epitope that doesnot include the whole sequence of the antigen or polypeptide from whichthe epitope was derived. Typically the “isolated” epitope does not haveattached thereto additional amino acids that result in a sequence thathas 100% identity with a native sequence. The native sequence can be asequence such as a tumor-associated antigen from which the epitope isderived.

[0048] A “lymphocyte” is a white blood cell derived from a stem cell ofthe primary lymphoid organs and which are responsible for mediating theimmune response. The term lymphocyte includes T cells, B cells andNatural Killer cells

[0049] “Major Histocompatibility Complex” or “MHC” is a cluster of genesthat plays a role in control of the cellular interactions responsiblefor physiologic immune responses. In humans, the MHC complex is alsoknown as the HLA complex. For a detailed description of the MHC and HLAcomplexes, see, Paul, FUNDAMENTAL IMMUNOLOGY, 3^(RD) ED., Raven Press,New York, 1993.

[0050] The term “motif” refers to the pattern of residues in a peptideof defined length, usually a peptide of from about 8 to about 13 aminoacids for a class I HLA motif and from about 6 to about 25 amino acidsfor a class II HLA motif, which is recognized by a particular HLAmolecule. Peptide motifs are typically different for each proteinencoded by each human HLA allele and differ in the pattern of theprimary and secondary anchor residues.

[0051] A “non-native” sequence or “construct” refers to a sequence thatis not found in nature (“non-naturally occurring”). Such sequencesinclude, e.g., peptides that are lipidated or otherwise modifed andpolyepitopic compositions that contain epitopes that are non contiguousin a native protein sequence.

[0052] The term “peptide” is used interchangeably with “oligopeptide” inthe present specification to designate a series of residues, typicallyL-amino acids, connected one to the other, typically by peptide bondsbetween the α-amino and carboxyl groups of adjacent amino acids. Thepreferred CTL-inducing peptides of the invention are 13 residues or lessin length and usually consist of between about 8 and about 11 residues,preferably 9 or 10 residues. The preferred HTL-inducing oligopeptidesare less than about 50 residues in length and usually consist of betweenabout 6 and about 30 residues, more usually between about 12 and 25, andoften between about 15 and 20 residues.

[0053] “pharmaceutically acceptable” refers to a generally non-toxic,inert, and/or physiologically compatible composition.

[0054] A “pharmaceutical excipient” comprises a material such as anadjuvant, a carrier, pH-adjusting and buffering agents, tonicityadjusting agents, wetting agents, preservative, and the like.

[0055] A “protective immune response” or “therapeutic immune response”refers to a CTL and/or an HTL response to an antigen derived from aninfectious agent or a tumor antigen, which prevents or at leastpartially arrests disease symptoms or progression. The immune responsemay also include an antibody response which has been facilitated by thestimulation of helper T cells.

[0056] The term “residue” refers to an amino acid or amino acid mimeticincorporated into an oligopeptide by an amide bond or amide bondmimetic.

[0057] “Synthetic peptide” refers to a peptide that is man-made usingsuch methods as chemical synthesis or recombinant DNA technology.

[0058] As used herein, a “vaccine” is a composition that contains one ormore peptides of the invention. There are numerous embodiments ofvaccines in accordance with the invention, such as by a cocktail of oneor more peptides; one or more epitopes of the invention comprised by apolyepitopic peptide; or nucleic acids that encode such peptides orpolypeptides, e.g., a minigene that encodes a monoepitopic orpolyepitopic peptide. The peptides or polypeptides can optionally bemodified, such as by lipidation, addition of targeting or othersequences. HLA class I-binding peptides of the invention can be admixedwith, or linked to, HLA class II-binding peptides, to facilitateactivation of both cytotoxic T lymphocytes and helper T lymphocytes.Vaccines can also comprise peptide-pulsed antigen presenting cells,e.g., dendritic cells.

[0059] The nomenclature used to describe peptide compounds follows theconventional practice wherein the amino group is presented to the left(the N-terminus) and the carboxyl group to the right (the C-terminus) ofeach amino acid residue. When amino acid residue positions are referredto in a peptide epitope they are numbered in an amino to carboxyldirection with position one being the position closest to the aminoterminal end of the epitope, or the peptide or protein of which it maybe a part In the formulae representing selected specific embodiments ofthe present invention, the amino- and carboxyl-terminal groups, althoughnot specifically shown, are in the form they would assume at physiologicpH values, unless otherwise specified. In the amino acid structureformulae, each residue is generally represented by standard three letteror single letter designations. The L-form of an amino acid residue isrepresented by a capital single letter or a capital first letter of athree-letter symbol, and the D-form for those amino acids having D-formsis represented by a lower case single letter or a lower case threeletter symbol. Glycine has no asymmetric carbon atom and is simplyreferred to as “Gly” or G. Symbols for the amino acids are shown below.Single Letter Symbol Three Letter Symbol Amino Acids A Ala Alanine C CysCysteine D Asp Aspartic Acid E Glu Glutamic Acid F Phe Phenylalanine GGly Glycine H His Histidine I Ile Isoleucine K Lys Lysine L Leu LeucineM Met Methionine N Asn Asparagine P Pro Proline Q Gln Glutamine R ArgArginine S Ser Serine T Thr Threonine V Val Valine W Trp Tryptophan YTyr Tyrosine

[0060] Stimulation of CTL and HTL Responses

[0061] The mechanism by which T cells recognize antigens has beendelineated during the past ten years. Based on our understanding of theimmune system we have developed efficacious peptide epitope vaccinecompositions that can induce a therapeutic or prophylactic immuneresponse to HPV in a broad population. For an understanding of the valueand efficacy of the claimed compositions, a brief review ofimmunology-related technology is provided.

[0062] A complex of an HLA molecule and a peptidic antigen acts as theligand recognized by HLA-restricted T cells (Buus, S. et al., Cell47:1071, 1986; Babbitt, B. P. et al., Nature 317:359, 1985; Townsend, A.and Bodmer, H., Annu. Rev. Immunol. 7:601, 1989; Germain, R. N., Annu.Rev. Immunol. 11:403, 1993). Through the study of single amino acidsubstituted antigen analogs and the sequencing of endogenously bound,naturally processed peptides, critical residues that correspond tomotifs required for specific binding to HLA antigen molecules have beenidentified (see, e.g., Southwood, et al, J. Immunol. 160:3363, 1998;Rammensee, et al., Immunogenetics 41:178, 1995; Rammensee et al,SYFPEITHI, access via web at:http://134.2.96.221/scripts.hlaserver.d11/home.htm; Sette, A. andSidney, J. Curr. Opin. Immunol. 10:478, 1998; Engelhard, V. H., Curr.Opin. Immunol. 6:13, 1994; Sette, A. and Grey, H. M., Curr. Opin.Immunol 4:79, 1992; Sinigaglia, F. and Hammer, J. Curr. Biol. 6:52,1994; Ruppert et al., Cell 74:929-937, 1993; Kondo et al., J. Immunol.155:4307-4312, 1995; Sidney et al., J. Immunol. 157:3480-3490, 1996;Sidney et al., Human Immunol 45:79-93, 1996, which are incorporatedherein by reference in their entireties, but also for their teachingregarding peptide binding motifs).

[0063] Furthermore, x-ray crystallographic analysis of HLA-peptidecomplexes has revealed pockets within the peptide binding cleft of HLAmolecules which accommodate, in an allele-specific mode, residues borneby peptide ligands; these residues in turn determine the HLA bindingcapacity of the peptides in which they are present. (See, e.g., Madden,D. R. Annu. Rev. Immunol. 13:587, 1995; Smith, et al., Immunity 4:203,1996; Fremont et al., Immunity 8:305, 1998; Stem et al., Structure2:245, 1994; Jones, E. Y. Curr. Opin. Immunol. 9:75, 1997; Brown, J. H.et al., Nature 364:33, 1993; Guo, H. C. et al., Proc. Natl. Acad. Sci.USA 90:8053, 1993; Guo, H. C. et al., Nature 360:364, 1992; Silver, M.L. et al., Nature 360:367, 1992; Matsumura, M. et al., Science 257:927,1992; Madden et al., Cell 70:1035, 1992; Fremont, D. H. et al., Science257:919, 1992; Saper, M. A., Bjorkman, P. J. and Wiley, D. C., J. Mol.Biol. 219:277, 1991, which are incorporated herein by reference in theirentireties, but also for their teaching regarding peptide binding.)

[0064] Accordingly, the definition of class I and class IIallele-specific HLA binding motifs, or class I or class II supermotifsallows identification of regions within a protein that have thepotential of binding particular HLA antigen(s).

[0065] Peptide Epitope Binding Motifs and Supermotifs

[0066] In the past few years evidence has accumulated to demonstratethat a large fraction of HLA class I and class II molecules can beclassified into a relatively few supertpes, each characterized bylargely overlapping peptide binding repertoires, and consensusstructures of the main peptide binding pockets.

[0067] For HLA molecule pocket analyses, the residues comprising the Band F pockets of HLA class I molecules as described in crystallographicstudies were analyzed (see, e.g., Guo, H. C. et al., Nature 360:364,1992; Saper, M. A., Bjorkman, P. J. and Wiley, D. C., J. Mol. Biol.219:277, 1991; Madden, D. R., Garboczi, D. N. and Wiley, D. C., Cell75:693, 1993; Parham, P., Adams, E. J., and Arnett, K. L., Immunol. Rev.143:141, 1995). In these analyses, residues 9, 45, 63, 66, 67, 70, and99 were considered to make up the B pocket; and the B pocket was deemedto determine the specificity for the amino acid residue in the secondposition of peptide ligands. Similarly, residues 77, 80, 81, and 116were considered to determine the specificity of the P pocket; the Fpocket was deemed to determine the specificity for the C-terminalresidue of a peptide ligand bound by the HLA class I molecule.

[0068] Through the study of single amino acid substituted antigenanalogs and the sequencing of endogenously bound, naturally processedpeptides, critical residues required for allele-specific binding to HLAmolecules have been identified. The presence of these residuescorrelates with binding affinity for HLA molecules. The identificationof motifs and/or supermotifs that correlate with high and intermediateaffinity binding is an important issue with respect to theidentification of immunogenic peptide epitopes for the inclusion in avaccine. Kast et al. (J. Immunol. 152:3904-3912, 1994) have shown thatmotif-bearing peptides account for 90% of the epitopes that bind toallele-specific HLA class I molecules. In this study all possiblepeptides of 9 amino acids in length and overlapping by eight amino acids(240 peptides), which cover the entire sequence of the E6 and E7proteins of human papillomavirus type 16, were evaluated for binding tofive allele-specific HLA molecules that are expressed at high frequencyamong different ethnic groups. This unbiased set of peptides allowed anevaluation of the predictive value of HLA class I motifs. From the setof 240 peptides, 22 peptides were identified that bound to anallele-specific HLA molecule with high or intermediate affinity. Ofthese 22 peptides, 20 (i.e. 91%) were motif-bearing. Thus, this studydemonstrates the value of motifs for the identification of peptideepitopes for inclusion in a vaccine: application of motif-basedidentification techniques eliminates screening of 90% of the potentialepitopes in a target antigen protein sequence.

[0069] Peptides of the present invention as set forth in Table 1 mayalso comprise epitopes that bind to MHC class II molecules. A greaterdegree of heterogeneity in both size and binding frame position of themotif, relative to the N and C termini of the peptide, exists for classII peptide ligands. This increased heterogeneity of HLA class II peptideligands is due to the structure of the binding groove of the HLA classII molecule which, unlike its class I counterpart, is open at both ends.Crystallographic analysis of HLA class II DRB*0101-peptide complexesshowed that the major energy of binding is contributed by peptideresidues complexed with complementary pockets on the DRB*0101 molecules.An important anchor residue engages the deepest hydrophobic pocket (see,e.g., Madden, D. R. Ann. Rev. Immunol. 13:587, 1995) and is referred toas position 1 (P1). P1 may represent the N-terminal residue of a classII binding peptide epitope, but more typically is flanked towards theN-terminus by one or more residues. Other studies have also pointed toan important role for the peptide residue in the 6^(th) position towardsthe C-terminus, relative to P1, for binding to various DR molecules.

[0070] Thus, peptides of the present invention as set forth in Table 1are identified by any one of several HLA-specific amino acid motifs. Ifthe presence of the motif corresponds to the ability to bind severalallele-specific HLA antigens, it is referred to as a supermotif. The HLAmolecules that bind to peptides that possess a particular amino acidsupermotif are collectively referred to as an HLA “supertype.”

[0071] Immune Response-Stimulating Peptide Analogs

[0072] In general, CTL and HTL responses are not directed against allpossible epitopes. Rather, they are restricted to a few “immunodominanf”determinants (Zinkernagel, et al, Adv. Immunol. 27:5159, 1979; Bennink,et al, J. Exp. Med. 168:19351939, 1988; Rawle, et al., J. Immunol.146:3977-3984, 1991). It has been recognized that immunodominance(Benacerraf, et al, Science 175:273-279, 1972) could be explained byeither the ability of a given epitope to selectively bind a particularHLA protein (determinant selection theory) (Vitiello, et al., J.Immunol. 131:1635, 1983); Rosenthal, et al., Nature 267:156-158, 1977),or to be selectively recognized by the existing TCR (T cell receptor)specificities (repertoire theory) (Klein, J., IMMUNOLOGY, THE SCIENCE OFSELFNONSELF DISCRIMINATION, John Wiley & Sons, New York pp. 270-310,1982). It has been demonstrated that additional factors, mostly linkedto processing events, can also play a key role in dictating, beyondstrict immunogenicity, which of the many potential determinants will bepresented as immunodominant (Sercarz, et al., Annu. Rev. Immunol.11:729-766, 1993).

[0073] The concept of dominance and subdominance is relevant toimmunotherapy of both infectious diseases and cancer. For example, inthe course of chronic viral disease, recruitment of subdominant epitopescan be important for successful clearance of the infection, especiallyif dominant CTL or HTL specificities have been inactivated by functionaltolerance, suppression, mutation of viruses and other mechanisms(Franco, et al., Curr. Opin. Immunol. 7:524-531, 1995; Zajac, et al., J.Exp. Med. 188:2205-2213, 1998). In the case of cancer and tumorantigens, CTLs recognizing at least some of the highest binding affinitypeptides might be functionally inactivated. Lower binding affinitypeptides are preferentially recognized at these times, and may thereforebe preferred in therapeutic or prophylactic anti-cancer vaccines.

[0074] In particular, it has been noted that a significant number ofepitopes derived from known non-viral tumor associated antigens (TAA)bind HLA class I with intermediate affinity (IC₅₀ in the 50-500 nMrange). For example, it has been found that 8 of 15 known TAA peptidesrecognized by tumor infiltrating lymphocytes (TIL) or CTL bound in the50-500 nM range. (These data are in contrast with estimates that 90% ofknown viral antigens were bound by HLA class I molecules with IC₅₀ of 50nM or less, while only approximately 10% bound in the 50-500 nM range(Sette, et al., J. Immunol., 153:558-5592, 1994). In the cancer settingthis phenomenon is probably due to elimination or functional inhibitionof the CTL recognizing several of the highest binding peptides,presumably because of T cell tolerization events.

[0075] Without intending to be bound by theory, it is believed thatbecause T cells to dominant epitopes may have been clonally deleted,selecting subdominant epitopes may allow existing T cells to berecruited, which will then lead to a therapeutic or prophylacticresponse. However, the binding of HLA molecules to subdominant epitopesis often less vigorous than to dominant ones. Accordingly, there is aneed to be able to modulate the binding affinity of particularimmunogenic epitopes for one or more HLA molecules, and thereby tomodulate the immune response elicited by the peptide, for example toprepare analog peptides which elicit a more vigorous response. Thisability would greatly enhance the usefulness of peptide-based vaccinesand therapeutic agents.

[0076] To ensure that an analog peptide, when used as a vaccine,actually elicits a CTL response to the native epitope in vivo (or, inthe case of class II epitopes, elicits helper T cells that cross-reactwith the wild type peptides), the analog peptide may be used to immunizeT cells in vitro from individuals of the appropriate HLA allele.Thereafter, the immunized cells' capacity to induce lysis of wild typepeptide sensitized target cells is evaluated. It will be desirable touse as antigen presenting cells, cells that have been either infected,or transfected with the appropriate genes, or, in the case of class IIepitopes only, cells that have been pulsed with whole protein antigens,to establish whether endogenously produced antigen is also recognized bythe relevant T cells.

[0077] Another embodiment for generating effective peptide analogsinvolves the substitution of residues that have an adverse impact onpeptide stability or solubility in, e.g., a liquid environment. Thissubstitution may occur at any position of the peptide epitope. Forexample, a cysteine (C) can be substituted out in favor of α-aminobutyric acid. Due to its chemical nature, cysteine has the propensity toform disulfide bridges and sufficiently alter the peptide structurallyso as to reduce binding capacity. Substituting α-amino butyric acid forC not only alleviates this problem, but actually improves binding andcrossbinding capability in certain instances (see, e.g. the review bySette et al., In: Persistent Viral Infections, Eds. R. Ahmed and I.Chen, John Wiley & Sons, England, 1999). Substitution of cysteine withα-amino butyric acid may occur at any residue of a peptide epitope, i.e.at either anchor or non-anchor positions.

[0078] Preparation of Peptide Epitopes

[0079] Peptides in accordance with the invention can be preparedsynthetically, by recombinant DNA technology or chemical synthesis, orfrom natural sources such as native tumors or pathogenic organisms.Peptide epitopes may be synthesized individually (monoepitopes) or aspolyepitopic peptides. Although the peptide will preferably besubstantially free of other naturally occurring host cell proteins andfragments thereof, in some embodiments the peptides may be syntheticallyconjugated to native fragments or particles.

[0080] The peptides in accordance with the invention and as set forth inExample 1 can be a variety of lengths, and either in their neutral(uncharged) forms or in forms which are salts. The peptides inaccordance with the invention are either free of modifications such asglycosylation, side chain oxidation, or phosphorylation; or they containthese modifications, subject to the condition that modifications do notdestroy the biological activity of the peptides as described herein.

[0081] The peptides of the invention can be prepared in a wide varietyof ways. For the preferred relatively short size, the peptides can besynthesized in solution or on a solid support in accordance withconventional techniques. Various automatic synthesizers are commerciallyavailable and can be used in accordance with known protocols. (See, forexample, Stewart & Young, SOLID PHASE PEPTIDE SYNTHESIS, 2D. ED., PierceChemical Co., 1984, which is incorporated herein by reference in itsentirety, but also for its teaching regarding peptide synthesismethods). Further, individual peptide epitopes can be joined usingchemical ligation to produce larger peptides that are still within thebounds of the invention.

[0082] Alternatively, recombinant DNA technology can be employed whereina nucleotide sequence which encodes an immunogenic peptide of interestis inserted into an expression vector, transformed or transfected intoan appropriate host cell and cultivated under conditions suitable forexpression. These procedures are generally known in the art, asdescribed generally in Sambrook et al., MOLECULAR CLONING, A LABORATORYMANUAL, Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (1989), whichis incorporated herein by reference in its entirety, but also for itsteaching regarding recombinant expression of peptides. Thus, recombinantpolypeptides which comprise one or more peptide sequences of theinvention can be used to present the appropriate T cell epitope.

[0083] The nucleotide coding sequence for peptide epitopes of thepreferred lengths contemplated herein can be synthesized by chemicaltechniques, for example, the phosphotriester method of Matteucci, etal., J. Am. Chem. Soc. 103:3185 (1981)), which is incorporated herein byreference in its entirety, but also for its teaching regardingrecombinant nucleic acids for expression of peptides. Peptide analogscan be made simply by substituting the appropriate and desired nucleicacid base(s) for those that encode the native peptide sequence;exemplary nucleic acid substitutions are those that encode an amino aciddefined by the motifs herein. The coding sequence can then be providedwith appropriate linkers and ligated into expression vectors commonlyavailable in the art, and the vectors used to transform suitable hoststo produce the desired fusion protein. A number of such vectors andsuitable host systems are now available. For expression of the fusionproteins, the coding sequence will be provided with operably linkedstart and stop codons, promoter and terminator regions and usually areplication system to provide an expression vector for expression in thedesired cellular host For example, promoter sequences compatible withbacterial hosts are provided in plasmids containing convenientrestriction sites for insertion of the desired coding sequence. Theresulting expression vectors are transformed into suitable bacterialhosts. Of course, yeast, insect or mammalian cell hosts may also beused, employing suitable vectors and control sequences.

[0084] It is often preferable that the peptide epitope be as small aspossible while still maintaining substantially all of the immunologicactivity of the native protein. When possible, it may be desirable tooptimize HLA class I binding peptide epitopes of the invention to alength of about 8 to about 13 amino acid residues, preferably 9 to 10.HLA class II binding peptide epitopes may be optimized to a length ofabout 6 to about 11 amino acids in length. Preferably, the peptideepitopes are commensurate in size with endogenously processedpathogen-derived peptides or tumor cell peptides that are bound to therelevant HLA molecules, however, the identification and preparation ofpeptides of other lengths can also be carried out using the techniquesdescribed herein.

[0085] In alternative embodiments, peptides of the invention can belinked as a polyepitopic peptide, or as a minigene that encodes amonoepitopic or polyepitopic peptide.

[0086] Assays to Detect T-Cell Responses

[0087] The HLA binding peptides identified in Example 1 can be testedfor the ability to elicit a T-cell response. The preparation andevaluation of motif-bearing peptides are described in PCT publicationsWO 94/20127 and WO 94/03205), which are incorporated herein by referencein their entirety, but also for their teaching regarding motif-bearingpeptides. Briefly, peptides comprising epitopes from a particularantigen are synthesized and tested for their ability to bind to theappropriate HLA proteins. These assays may involve evaluating thebinding of a peptide of the invention to purified HLA class I moleculesin relation to the binding of a radioiodinated reference peptide.Alternatively, cells expressing empty class I molecules (i.e. lackingpeptide therein) may be evaluated for peptide binding byimmunofluorescent staining and flow microfluorimetry. Other assays thatmay be used to evaluate peptide binding include peptide-dependent classI assembly assays and/or the inhibition of CTL recognition by peptidecompetition. Those peptides that bind to the class I molecule, typicallywith an affinity of 500 nM or less, are further evaluated for theirability to serve as targets for CTLs derived from infected or immunizedindividuals, as well as for their capacity to induce primary in vitro orin vivo CTL responses that can give rise to CTL populations capable ofreacting with selected target cells associated with a disease.Corresponding assays are used for evaluation of HLA class II bindingpeptides. HLA class II motif-bearing peptides that are shown to bind,typically at an affinity of 1000 nM or less, are further evaluated forthe ability to stimulate HTL responses.

[0088] Conventional assays utilized to detect T cell responses includeproliferation assays, lymphokine secretion assays, direct cytotoxicityassays, and limiting dilution assays. For example, antigen-presentingcells that have been incubated with a peptide can be assayed for theability to induce CTL responses in responder cell populations.Antigen-presenting cells can be normal cells such as peripheral bloodmononuclear cells or dendritic cells. Alternatively, mutant non-humanmammalian cell lines that are deficient in their ability to load class Imolecules with internally processed peptides and that have beentransfected with the appropriate human class I gene, may be used to testfor the capacity of the peptide to induce in vitro primary CTLresponses.

[0089] Peripheral blood mononuclear cells (PBMCs) may be used as theresponder cell source of CTL precursors. The appropriateantigen-presenting cells are incubated with peptide, after which thepeptide-loaded antigen-presenting cells are then incubated with theresponder cell population under optimized culture conditions. PositiveCTL activation can be determined by assaying the culture for thepresence of CTLs that kill radio-labeled target cells, both specificpeptide-pulsed targets as well as target cells expressing endogenouslyprocessed forms of the antigen from which the peptide sequence wasderived.

[0090] More recently, a method has been devised which allows directquantification of antigen-specific T cells by staining withFluorescein-labelled HLA tetrameric complexes (Altman, J. D. et al.,Proc. Natl. Acad. Sci. USA 90:10330,1993; Altman, J. D. et al., Science274:94, 1996), which are incorporated herein by reference in theirentirety, but also for their teaching regarding quantification of Tcells). Other relatively recent technical developments include stainingfor intracellular lymphokines, and interferon release assays or ELISPOTassays. Tetramer staining, intracellular lympholine staining and ELISPOTassays all appear to be at least 10-fold more sensitive than moreconventional assays (Lalvani, A. et al., J. Exp. Med. 186:859, 1997;Dunbar, P. R. et al., Curr. Biol. 8:413, 1998; Murali-Krishna, K. etal., Immunity 8:177, 1998, which are incorporated herein by reference intheir entirety, but also for their teaching regarding quantification ofT cells).

[0091] HTL activation may also be assessed using such techniques knownto those in the art such as T cell proliferation and secretion oflymphokines, e.g. IL-2 (see, e.g. Alexander et al., Immunity 1:751-761,1994).

[0092] Alternatively, immunization of HLA transgenic mice can be used todetermine immunogenicity of peptide epitopes. Several transgenic mousemodels including mice with human A2.1, A11 (which can additionally beused to analyze HLA-A3 epitopes), and B7 alleles have been characterizedand others (e.g., transgenic mice for HLA-A1 and A24) are beingdeveloped. HLA-DR1 and HLA-DR3 mouse models have also been developed.Additional transgenic mouse models with other HLA alleles may begenerated as necessary. Mice may be immunized with peptides emulsifiedin Incomplete Freund's Adjuvant and the resulting T cells tested fortheir capacity to recognize peptide-pulsed target cells and target cellstransfected with appropriate genes. CTL responses may be analyzed usingcytotoxicity assays described above. Similarly, HTL responses may beanalyzed using such assays as T cell proliferation or secretion oflymphokines.

[0093] Use of Peptide Epitopes as Diagnostic Agents and for EvaluatingImmune Responses

[0094] In one embodiment of the invention, HLA class I and class IIbinding peptides as described in Example 1 can be used as reagents toevaluate an immune response. The immune response to be evaluated can beinduced by using as an immunogen any agent that may result in theproduction of antigen-specific CTLs or HTLs that recognize and bind tothe peptide epitope(s) to be employed as the reagent. The peptidereagent need not be used as the immunogen. Assay systems that can beused for such an analysis include relatively recent technicaldevelopments such as tetramers, staining for intracellular lymphokinesand interferon release assays, or ELISPOT assays.

[0095] For example, a peptide of the invention may be used in a tetramerstaining assay to assess peripheral blood mononuclear cells for thepresence of antigen-specific CTLs following exposure to a tumor cellantigen or an immunogen. The HLA-tetrameric complex is used to directlyvisualize antigen-specific CTLs (see, e.g., Ogg et al, Science279:2103-2106, 1998; and Altman et al., Science 174:94-96, 1996, whichare incorporated herein by reference in their entirety, but also fortheir teaching regarding quantification of T cells) and determine thefrequency of the antigen-specific CTL population in a sample ofperipheral blood mononuclear cells. A tetramer reagent using a peptideof the invention may be generated as follows: A peptide that binds to anHLA molecule is refolded in the presence of the corresponding HLA heavychain and β₂-microglobulin to generate a trimolecular complex. Thecomplex is biotinylated at the carboxyl terminal end of the heavy chainat a site that was previously engineered into the protein. Tetramerformation is then induced by the addition of streptavidin. By means offluorescently labeled streptavidin, the tetramer can be used to stainantigen-specific cells. The cells may then be identified, for example,by flow cytometry. Such an analysis may be used for diagnostic orprognostic purposes. Cells identified by the procedure can also be usedfor therapeutic purposes.

[0096] Peptides of the invention may also be used as reagents toevaluate immune recall responses. (see, e.g., Bertoni et al., J. Clin.Invest. 100:503-513, 1997 and Penna et al., J. Exp. Med. 174:1565-1570,1991, which are incorporated herein by reference in their entirety, butalso for their teaching regarding quantification of T cells.) Forexample, patient PBMC samples from individuals with HPV infection may beanalyzed for the presence of antigen-specific CTLs or HTLs usingspecific peptides. A blood sample containing mononuclear cells may beevaluated by cultivating the PBMCs and stimulating the cells with apeptide of the invention. After an appropriate cultivation period, theexpanded cell population may be analyzed, for example, for cytotoxicactivity (CTL) or for HTL activity.

[0097] The peptides may also be used as reagents to evaluate theefficacy of a vaccine. PBMCs obtained from a patient vaccinated with animmunogen may be analyzed using, for example, either of the methodsdescribed above. The patient is HLA typed, and peptide epitope reagentsthat recognize the allele-specific molecules present in that patient areselected for the analysis. The immunogenicity of the vaccine isindicated by the presence of epitope-specific CTLs and/or HTLs in thePBMC sample.

[0098] The peptides of the invention may also be used to makeantibodies, using techniques well known in the art (see, e.g. CURRENTPROTOCOLS IN IMMUNOLOGY, Wiley/Greene, N.Y.; and Antibodies A LaboratoryManual, Harlow and Lane, Cold Spring Harbor Laboratory Press, 1989,which is incorporated herein by reference in its entirety, but also forits teaching regarding antibody preparation), which may be useful asreagents to diagnose or monitor cancer. Such antibodies include thosethat recognize a peptide in the context of an HLA molecule, i.e.,antibodies that bind to a peptide-MHC complex.

[0099] Vaccine Compositions

[0100] Vaccines and methods of preparing vaccines that contain animmunogenically effective amount of one or more peptides as describedherein are further embodiments of the invention. Once appropriatelyimmunogenic epitopes have been defined, they can be sorted and deliveredby various means, herein referred to as “vaccine” compositions. Suchvaccine compositions can include, for example, lipopeptides (e.g.,Vitiello, A. et al., J. Clin. Invest. 95:341, 1995), peptidecompositions encapsulated in poly(DL-lactideco-glycolide) (“PLG”)microspheres (see, e.g., Eldridge, et al., Molec. Immunol. 28:287-294,1991: Alonso et al., Vaccine 12:299-306, 1994; Jones et al., Vaccine13:675-681, 1995), peptide compositions contained in immune stimulatingcomplexes (ISCOMS) (see, e.g. Takahashi et al., Nature 344:873-875,1990; Hu et al., Clin Exp Immunol. 113:235-243, 1998), multiple antigenpeptide systems (MAPs) (see e.g., Tam, J. P., Proc. Natl. Acad. Sci.U.S.A. 85:5409-5413, 1988; Tam, J. P., J. Immunol. Methods 196:17-32,1996), viral delivery vectors (Perkus, M. E. et al., In: Concepts invaccine development, Kaufmann, S. H. E., ed., p. 379, 1996; Chakrabarti,S. et al., Nature 320:535, 1986; Hu, S. L. et al., Nature 320:537, 1986;Kieny, M.-P. et al., AIDS Bio/Technology 4:790, 1986; Top, F. H. et al,J. Infect. Dis. 124:148, 1971; Chanda, P. K. et al., Virology 175:535,1990), particles of viral or synthetic origin (e.g., Kofler, N. et al.,J. Immunol. Methods. 192:25, 1996; Eldridge, J. H. et al., Sem. Hematol30:16, 1993; Falo, L. D., Jr. et al., Nature Med. 7:649, 1995),adjuvants (Warren, H. S., Vogel, F. R., and Chedid, L. A. Annu. Rev.Immunol. 4:369, 1986; Gupta, R. K. et al., Vaccine 11:293, 1993),liposomes (Reddy, R. et al., J. Immunol. 148:1585, 1992; Rock, K. L.,Immunol. Today 17:131, 1996), or, naked or particle absorbed cDNA(Ulmer, J. B. et al., Science 259:1745, 1993; Robinson, H. L., Hunt, L.A., and Webster, R. G., i Vaccine 11:957, 1993; Shiver, J. W. et al.,In: Concepts in vaccine development, Kaufmann, S. H. E., ed., p. 423,1996; Cease, K. B., and Berzofsky, J. A., Annu. Rev. Immunol. 12:923,1994 and Eldridge, J. H. et al., Sem. Hematol. 30:16, 1993, which areincorporated herein by reference in their entirety, but also for theirteaching regarding vaccine compositions). Toxin-targeted deliverytechnologies, also known as receptor mediated targeting, such as thoseof Avant Immunotherapeutics, Inc. (Needham, Mass.) may also be used.

[0101] Vaccines of the invention include nucleic acid-mediatedmodalities. DNA or RNA encoding one or more of the peptides of theinvention can also be administered to a patient. This approach isdescribed, for instance, in Wolff et. al., Science 247:1465 (1990) aswell as U.S. Pat. Nos. 5,580,859; 5,589,466; 5,804,566; 5,739,118;5,736,524; 5,679,647; WO 98/04720 (which are incorporated herein byreference in their entirety, but also for their teaching regardingvaccine compositions); and in more detail below. Examples of DNA-baseddelivery technologies include “naked DNA”, facilitated (bupivicaine,polymers, peptide-mediated) delivery, cationic lipid complexes, andparticle-mediated (“gene gun”) or pressure-mediated delivery (see, e.g.,U.S. Pat. No. 5,922,687, which is incorporated herein by reference inits entirety, but also for its teaching regarding DNA vaccines).

[0102] For therapeutic or prophylactic immunization purposes, thepeptides of the invention can also be expressed by viral or bacterialvectors. Examples of expression vectors include attenuated viral hosts,such as vaccinia or fowlpox. As an example of this approach, vacciniavirus is used as a vector to express nucleotide sequences that encodethe peptides of the invention. Upon introduction into a host bearing atumor, the recombinant vaccinia virus expresses the immunogenic peptide,and thereby elicits a host CTL and/or HTL response. Vaccinia vectors andmethods useful in immunization protocols are described in, e.g., U.S.Pat. No. 4,722,848. Another vector is BCG (Bacille Calmette Guerin). BCGvectors are described in Stover et al., Nature 351:456-460 (1991). Awide variety of other vectors useful for therapeutic administration orimmunization of the peptides of the invention, e.g. adeno andadeno-associated virus vectors, retroviral vectors, Salmonella typhivectors, detoxified anthrax toxin vectors, and the like, will beapparent to those skilled in the art from the description herein.

[0103] Furthermore, vaccines in accordance with the invention encompasscompositions of one or more of the claimed peptide(s) as described inExample 1. A peptide can be present in a vaccine individually.Alternatively, the peptide can exist as a homopolymer comprisingmultiple copies of the same peptide, or as a heteropolymer of variouspeptides. Polymers have the advantage of increased immunologicalreaction and, where different peptide epitopes are used to make up thepolymer, the additional ability to induce antibodies and/or CTLs thatreact with different antigenic determinants of the pathogenic organismor tumor-related peptide targeted for an immune response. Thecomposition can be a naturally occurring region of an antigen or can beprepared, e.g., recombinantly or by chemical synthesis.

[0104] Carriers that can be used with vaccines of the invention are wellknown in the art, and include, e.g., thyroglobulin, albumins such ashuman serum albumin, tetanus toxoid, polyamino acids such as polyL-lysine, poly L-glutamic acid, influenza, hepatitis B virus coreprotein, and the like. The vaccines can contain a physiologicallytolerable (i.e., acceptable) diluent such as water, or saline,preferably phosphate buffered saline. The vaccines also typicallyinclude an adjuvant. Adjuvants such as incomplete Freund's adjuvant,aluminum phosphate, aluminum hydroxide, or alum are examples ofmaterials well known in the art. Additionally, as disclosed herein, CTLresponses can be primed by conjugating peptides of the invention tolipids, such as tripalmitoyl-S-glycerylcysteinlyseryl-serine (P₃CSS).

[0105] Upon immunization with a peptide composition in accordance withthe invention, via injection, aerosol, oral, transdermal, transmucosal,intrapleural, intrathecal, or other suitable routes, the immune systemof the host responds to the vaccine by producing large amounts of CTLsand/or HTLs specific for the desired antigen. Consequently, the hostbecomes at least partially immune to later infection, or at leastpartially resistant to developing an ongoing chronic infection, orderives at least some therapeutic benefit when the antigen wastumor-associated.

[0106] In some embodiments it may be desirable to combine the class Ipeptide components with components that induce or facilitateneutralizing antibody responses to the target antigen of interest,particularly to viral envelope antigens. A preferred embodiment of sucha composition comprises class I and class II epitopes in accordance withthe invention.

[0107] A vaccine of the invention can also include antigen-presentingcells, such as dendritic cells, as a vehicle to present peptides of theinvention. Vaccine compositions can be created in vitro, followingdendritic cell mobilization and harvesting, whereby loading of dendriticcells occurs in vitro. For example, dendritic cells are transfected,e.g., with a minigene in accordance with the invention. The dendriticcell can then be administered to a patient to elicit immune responses invivo.

[0108] Antigenic peptides are used to elicit a CTL and/or HTL responseex vivo, as well. The resulting CTL or HTL cells, can be used to treattumors in patients that do not respond to other conventional forms oftherapy, or will not respond to a therapeutic vaccine peptide or nucleicacid in accordance with the invention. Ex vivo CTL or HTL responses to aparticular tumor-associated antigen are induced by incubating in tissueculture the patients, or genetically compatible, CTL or HTL precursorcells together with a source of antigen-presenting cells (APC), such asdendritic cells, and the appropriate immunogenic peptide. After anappropriate incubation time (typically about 7-28 days), in which theprecursor cells are activated and expanded into effector cells, thecells are infused back into the patient, where they will destroy (CTL)or facilitate destruction (HTL) of their specific target cell (aninfected cell or a tumor cell). Transfected dendritic cells may also beused as antigen presenting cells.

[0109] The vaccine compositions of the invention can also be used incombination with antiviral drugs such as interferon-α, or othertreatments for viral infection.

[0110] Exemplary epitopes that may be utilized in a vaccine to treat orprevent HPV infection are set out in Example 1 and Table 1.

[0111] If a polyepitopic protein is created, or when creating aminigene, an objective is to generate the smallest peptide thatencompasses the epitopes of interest. This principle is similar, if notthe same as that employed when selecting a peptide comprising nestedepitopes. However, with an artificial polyepitopic peptide, the sizeminimization objective is balanced against the need to integrate anyspacer sequences between epitopes in the polyepitopic protein. Spaceramino acid residues can be introduced to avoid junctional epitopes (anepitope recognized by the immune system, not present in the targetantigen, and only created by the man-made juxtaposition of epitopes), orto facilitate cleavage between epitopes and thereby enhance epitopepresentation. Junctional epitopes are generally to be avoided becausethe recipient may generate an immune response to that non-nativeepitope. Of particular concern is a junctional epitope that is a“dominant epitope.” A dominant epitope may lead to such a zealousresponse that immune responses to other epitopes are diminished orsuppressed.

[0112] Specific embodiments of the polyepitopic compositions of thepresent invention include a pharmaceutical composition comprising apharmaceutically acceptable carrier and combination of motif-bearingpeptides that are immunologically cross-reactive with peptides of HPV,wherein at least one of the peptides bears a motif of SEQ ID NO. 1, anda second peptide selected from Table 1.

[0113] Minigene Vaccines

[0114] A number of different approaches are available which allowsimultaneous delivery of multiple epitopes. Nucleic acids encoding thepeptides of the invention are a particularly useful embodiment of theinvention. Epitopes for inclusion in a minigene are preferably selectedaccording to the guidelines set forth in the previous section. Apreferred means of administering nucleic acids encoding the peptides ofthe invention uses minigene constructs encoding a peptide comprising oneor multiple epitopes of the invention.

[0115] The use of multi-epitope minigenes is described below and in,e.g. An, L. and Whitton, J. L., J. Virol. 71:2292, 1997; Thomson, S. A.et al., J. Immunol. 157:822, 1996; Whitton, J. L. et al., J. Virol.67:348, 1993; Hanke, R. et al., Vaccine 16:426, 1998, which areincorporated herein by reference in their entirety, but also for theirteaching regarding multi-epitope minigene vaccine compositions. Forexample, a multi-epitope DNA plasmid encoding SEQ ID NO. 1 and anendoplasmic reticulum-translocating signal sequence can be engineered.

[0116] The immunogenicity of a multi-epitopic minigene can be tested intransgenic mice to evaluate the magnitude of CTL induction responsesagainst the epitopes tested. Further, the immunogenicity of DNA-encodedepitopes in vivo can be correlated with the in vitro responses ofspecific CTL lines against target cells transfected with the DNAplasmid. Thus, these experiments can show that the minigene serves toboth: 1.) generate a CTL response and 2.) that the induced CTLsrecognized cells expressing the encoded epitopes.

[0117] For example, to create a DNA sequence encoding the selectedepitopes (minigene) for expression in human cells, the amino acidsequences of the epitopes may be reverse translated. A human codon usagetable can be used to guide the codon choice for each amino acid. Theseepitope-encoding DNA sequences may be directly adjoined, so that whentranslated, a continuous polypeptide sequence is created. To optimizeexpression and/or immunogenicity, additional elements can beincorporated into the minigene design. Examples of amino acid sequencesthat can be reverse translated and included in the minigene sequenceinclude: HLA class I epitopes, HLA class II epitopes, a ubiquitinationsignal sequence, and/or an endoplasmic reticulum targeting signal. Inaddition, HLA presentation of CTL and HTL epitopes may be improved byincluding synthetic (e.g. poly-alanine) or naturally-occurring flankingsequences adjacent to the CTL or HTL epitopes; these larger peptidescomprising the epitope(s) are within the scope of the invention.

[0118] The minigene sequence may be converted to DNA by assemblingoligonucleotides that encode the plus and minus strands of the minigene.Overlapping oligonucleotides (30-100 bases long) may be synthesized,phosphorylated, purified and annealed under appropriate conditions usingwell known techniques. The ends of the oligonucleotides can be joined,for example, using T4 DNA ligase. This synthetic minigene, encoding theepitope polypeptide, can then be cloned into a desired expressionvector.

[0119] Standard regulatory sequences well known to those of skill in theart are preferably included in the vector to ensure expression in thetarget cells. Several vector elements are desirable: a promoter with adown-stream cloning site for minigene insertion; a polyadenylationsignal for efficient transcription termination; an E. coli origin ofreplication; and an E. coli selectable marker (e.g. ampicillin orkanamycin resistance). Numerous promoters can be used for this purpose,e.g., the human cytomegalovirus (HCMV) promoter. See, e.g., U.S. Pat.Nos. 5,580,859 and 5,589,466 for other suitable promoter sequences.

[0120] Additional vector modifications may be desired to optimizeminigene expression and immunogenicity. In some cases, introns arerequired for efficient gene expression, and one or more synthetic ornaturally-occurring introns could be incorporated into the transcribedregion of the minigene. The inclusion of mRNA stabilization sequencesand sequences for replication in mammalian cells may also be consideredfor increasing minigene expression.

[0121] Once an expression vector is selected, the minigene is clonedinto the polylinker region downstream of the promoter. This plasmid istransformed into an appropriate E. coli strain, and DNA is preparedusing standard techniques. The orientation and DNA sequence of theminigene, as well as all other elements included in the vector, areconfirmed using restriction mapping and DNA sequence analysis. Bacterialcells harboring the correct plasmid can be stored as a master cell bankand a working cell bank.

[0122] In addition, immunostimulatory sequences (ISSs or CpGs) appear toplay a role in the immunogenicity of DNA vaccines. These sequences maybe included in the vector, outside the minigene coding sequence, ifdesired to enhance immunogenicity.

[0123] In some embodiments, a bi-cistronic expression vector whichallows production of both the minigene-encoded epitopes and a secondprotein (included to enhance or decrease immunogenicity) can be used.Examples of proteins or polypeptides that could beneficially enhance theimmune response if co-expressed include cytokines (e.g., IL-2, IL-12,GM-CSF), cytokine-inducing molecules (e.g., LeIF), costimulatorymolecules, or for HTL responses. Helper (HTL) epitopes can be joined tointracellular targeting signals and expressed separately from expressedCTL epitopes; this allows direction of the HTL epitopes to a cellcompartment different than that of the CTL epitopes. If required, thiscould facilitate more efficient entry of HTL epitopes into the HLA classII pathway, thereby improving HTL induction. In contrast to HTL or CTLinduction, specifically decreasing the immune response by co-expressionof immunosuppressive molecules (e.g. TGF-β) may be beneficial in certaindiseases.

[0124] Therapeutic quantities of plasmid DNA can be produced forexample, by fermentation in E. coli, followed by purification. Aliquotsfrom the working cell bank are used to inoculate growth medium, andgrown to saturation in shaker flasks or a bioreactor according to wellknown techniques. Plasmid DNA can be purified using standardbioseparation technologies such as solid phase anion-exchange resinssupplied by QIAGEN, Inc. (Valencia, Calif.). If required, supercoiledDNA can be isolated from the open circular and linear forms using gelelectrophoresis or other methods.

[0125] Purified plasmid DNA can be prepared for injection using avariety of formulations. The simplest of these is reconstitution oflyophilized DNA in sterile phosphate-buffer saline (PBS). This approach,known as “naked DNA,” is currently being used for intramuscular (IM)administration in clinical trials. To maximize the immunotherapeuticeffects of minigene DNA vaccines, an alternative method for formulatingpurified plasmid DNA may be desirable. A variety of methods have beendescribed, and new techniques may become available. Cationic lipids canalso be used in the formulation (see, e.g., as described by WO 93/24640;Mannino & Gould-Fogerite, BioTechniques 6(7): 682 (1988); U.S. Pat. No.5,279,833; WO 91/06309; and Felgner, et al., Proc. Nat'l Acad. Sci. USA84:7413 (1987), which are incorporated herein by reference in theirentirety, but also for their teaching regarding DNA vaccinecompositions. In addition, glycolipids, fusogenic liposomes, peptidesand compounds referred to collectively as protective, interactive,non-condensing compounds (PINC) could also be complexed to purifiedplasmid DNA to influence variables such as stability, intramusculardispersion, or trafficking to specific organs or cell types.

[0126] Target cell sensitization can be used as a functional assay forexpression and HLA class I presentation of minigene-encoded CTLepitopes. For example, the plasmid DNA is introduced into a mammaliancell line that is suitable as a target for standard CTL chromium releaseassays. The transfection method used will be dependent on the finalformulation. Electroporafion can be used for “naked” DNA, whereascationic lipids allow direct in vitro transfection. A plasmid expressinggreen fluorescent protein (GFP) can be co-transfected to allowenrichment of transfected cells using fluorescence activated cellsorting (FACS). These cells are then chromium-51 (⁵¹Cr) labeled and usedas target cells for epitope-specific CTL lines; cytolysis, detected by⁵¹Cr release, indicates both production of, and HLA presentation of,minigene-encoded CTL epitopes. Expression of HTL epitopes may beevaluated in an analogous manner using assays to assess HTL activity.

[0127] In vivo immunogenicity is a second approach for functionaltesting of minigene DNA formulations. Transgenic mice expressingappropriate human HLA proteins are immunized with the DNA product. Thedose and route of administration are formulation dependent (e.g., IM forDNA in PBS, intraperitoneal (IP) for lipid-complexed DNA). Twenty-onedays after immunization, splenocytes are harvested and restimulated for1 week in the presence of peptides encoding each epitope being tested.Thereafter, for CTL effector cells, assays are conducted for cytolysisof peptide-loaded, ⁵¹Cr-labeled target cells using standard techniques.Lysis of target cells that were sensitized by HLA loaded with peptideepitopes, corresponding to minigene-encoded epitopes, demonstrates DNAvaccine function for in vivo induction of CTLs. Immunogenicity of HTLepitopes is evaluated in transgenic mice in an analogous manner.

[0128] Alternatively, the nucleic acids can be administered usingballistic delivery as described, for instance, in U.S. Pat. No.5,204,253, which is incorporated herein by reference in its entirety,but also for its teaching regarding ballistic DNA delivery. Using thistechnique, particles comprised solely of DNA are administered. In afurther alternative embodiment, DNA can be adhered to particles, such asgold particles.

[0129] Combinations of CTL Peptides with Helper Peptides

[0130] Vaccine compositions comprising the peptides of the presentinvention, or analogs thereof, which have immunostimulatory activity maybe modified to provide desired attributes, such as improved serum halflife, or to enhance immunogenicity.

[0131] For instance, the ability of the peptide KLPDLCTEL(SEQ ID NO:1)to induce CTL activity can be enhanced by linking the peptide to asequence which contains at least one epitope that is capable of inducinga T helper cell response such as DRAHYNI (SEQ ID NO. 2).

[0132] Particularly preferred CTL epitope/HTL epitope conjugates arelinked by a spacer molecule. The spacer is typically comprised ofrelatively small, neutral molecules, such as amino acids or amino acidmimetics, which are substantially uncharged under physiologicalconditions. The spacers are typically selected from, e.g., Ala, Gly, orother neutral spacers of nonpolar amino acids or neutral polar aminoacids. It will be understood that the optionally present spacer need notbe comprised of the same residues and thus may be a hetero- orhomo-oligomer. When present, the spacer will usually be at least one ortwo residues, more usually three to six residues. Alternatively, the CTLpeptide may be linked to the T helper peptide without a spacer. Theamino terminus of either the immunogenic peptide or the T helper peptidemay be acylated.

[0133] The HTL peptide epitope can also be modified to alter itsbiological properties. For example, peptides comprising HTL epitopes cancontain D-amino acids to increase their resistance to proteases and thusextend their serum half-life. Also, the epitope peptides of theinvention can be conjugated to other molecules such as lipids, proteinsor sugars, or any other synthetic compounds, to increase theirbiological activity. Specifically, the T helper peptide can beconjugated to one or more palmitic acid chains at either the amino orcarboxyl termini.

[0134] In some embodiments it may be desirable to include in thepharmaceutical compositions of the invention at least one componentwhich primes cytotoxic T lymphocytes. Lipids have been identified asagents capable of priming CTL in vivo against viral antigens. Forexample, palmitic acid residues can be attached to the 6-and α-aminogroups of a lysine residue and then linked, e.g., via one or morelinking residues such as Gly, Gly-Gly-, Ser, Ser-Ser, or the like, to animmunogenic peptide. The lipidated peptide can then be administeredeither directly in a micelle or particle, incorporated into a liposome,or emulsified in an adjuvant, e.g., incomplete Freund's adjuvant In apreferred embodiment, a particularly effective immunogenic comprisespalmitic acid attached to ε- and α-amino groups of Lys, which isattached via linkage, e.g., Ser-Ser, to the amino terminus of theimmunogenic peptide.

[0135] As another example of lipid priming of CTL responses, E. colilipoproteins, such as tripalmitoyl-S-glycerylcysteinlyseryl-serine(P₃CSS) can be used to prime virus specific CTL when covalently attachedto an appropriate peptide. (See, e.g., Deres, et al., Nature 342:561,1989). Peptides of the invention can be coupled to P₃CSS, for example,and the lipopeptide administered to an individual to specifically primea CTL response to the target antigen. Moreover, because the induction ofneutralizing antibodies can also be primed with P₃CSS-conjugatedepitopes, two such compositions can be combined to more effectivelyelicit both humoral and cell-mediated responses to infection.

[0136] As noted herein, additional amino acids can be added to thetermini of a peptide to provide for ease of linking peptides one toanother, for coupling to a carrier support or larger peptide, formodifying the physical or chemical properties of the peptide oroligopeptide, or the like. Amino acids such as tyrosine, cysteine,lysine, glutamic or aspartic acid, or the like, can be introduced at theC- or N-terminus of the peptide or oligopeptide, particularly class Ipeptides. However, it is to be noted that modification at the carboxylterminus of a CTL epitope may, in some cases, alter bindingcharacteristics of the peptide. In addition, the peptide or oligopeptidesequences can differ from the natural sequence by being modified byterminal-NH₂ acylation, e.g., by alkanoyl (C₁—C₂₀) or thioglycolylacetylation, terminal-carboxyl amidation, e.g., ammonia, methylamine,etc. In some instances these modifications may provide sites for linkingto a support or other molecule.

[0137] Vaccine Compositions Comprising Dendritic Cells (DC) Pulsed withCTL and/or HTL Peptides

[0138] An embodiment of a vaccine composition in accordance with theinvention comprises ex vivo administration of peptides to PBMC, orisolated DC therefrom, from the patient's blood. A pharmaceutical tofacilitate harvesting of DC can be used, such as GM-CSF/IL4. Afterpulsing the DC with peptides and prior to reinfusion into patients, theDC are washed to remove unbound peptides. In this embodiment, a vaccinecomprises peptide-pulsed DCs which present the pulsed peptide epitopescomplexed with HLA molecules on their surfaces. The vaccine is thenadministered to the patient.

[0139] Administration of Vaccines for Therapeutic or ProphylacticPurposes

[0140] The peptides of the present invention and pharmaceutical andvaccine compositions of the invention are useful for administration tomammals, particularly humans, to treat and/or prevent HPV infection.Vaccine compositions containing the peptides of the invention areadministered to a patient infected with HPV or to an individualsusceptible to, or otherwise at risk for, HPV infection to elicit animmune response against HPV antigens and thus enhance the patient's ownimmune response capabilities. In therapeutic applications, peptideand/or nucleic acid compositions are administered to a patient in anamount sufficient to elicit an effective CTL and/or HTL response to thevirus antigen and to cure or at least partially arrest or slow symptomsand/or complications. An amount adequate to accomplish this is definedas “therapeutically effective dose.” Amounts effective for this use willdepend on, e.g., the particular composition administered, the manner ofadministration, the stage and severity of the disease being treated, theweight and general state of health of the patient, and the judgment ofthe prescribing physician.

[0141] The vaccine compositions of the invention may also be used purelyas prophylactic agents. Generally the dosage for an initial prophylacticimmunization generally occurs in a unit dosage range where the lowervalue is about 1, 5, 50, 500, or 1000 μg of peptide and the higher valueis about 10,000; 20,000; 30,000; or 50,000 μg. Dosage values for a humantypically range from about 500 μg to about 50,000 μg per 70 kilogrampatient. This is followed by boosting dosages of between about 1.0 μg toabout 50,000 μg of peptide administered at defined intervals from aboutfour weeks to six months after the initial administration of vaccine.The immunogenicity of the vaccine may be assessed by measuring thespecific activity of CTL and HTL obtained from a sample of the patient'sblood.

[0142] As noted above, peptides comprising CTL and/or HTL epitopes ofthe invention induce immune responses when presented by HLA moleculesand contacted with a CTL or HTL specific for an epitope comprised by thepeptide. The manner in which the peptide is contacted with the CTL orHTL is not critical to the invention. For instance, the peptide can becontacted with the CTL or HTL either in vivo or in vitro. If thecontacting occurs in vivo, the peptide itself can be administered to thepatient, or other vehicles, e.g., DNA vectors encoding one or morepeptides, viral vectors encoding the peptide(s), liposomes and the like,can be used, as described herein. When the peptide is contacted invitro, the vaccinating agent can comprise a population of cells, e.g.,peptide-pulsed dendritic cells, or TAA-specific CTLs, which have beeninduced by pulsing antigen-presenting cells in vitro with the peptide.Such a cell population is subsequently administered to a patient in atherapeutically effective dose.

[0143] The peptides or DNA encoding them can be administeredindividually or as fusions of one or more peptide sequences.

[0144] For pharmaceutical compositions, the immunogenic peptides of theinvention, or DNA encoding them, are generally administered to anindividual already infected with HPV. The peptides or DNA encoding themcan be administered individually or as fusions of one or more peptidesequences. Those in the incubation phase or the acute phase of infectioncan be treated with the immunogenic peptides separately or inconjunction with other treatments, as appropriate.

[0145] For therapeutic use, administration should generally begin at thefirst diagnosis of HPV infection. This is followed by boosting dosesuntil at least symptoms are substantially abated and for a periodthereafter. In chronic infection, loading doses followed by boostingdoses may be required.

[0146] The peptide or other compositions used for the treatment orprophylaxis of HPV infection can be used, e.g., in persons who have notmanifested symptoms of disease but who act as a disease vector. In thiscontext, it is generally important to provide an amount of the peptideepitope delivered by a mode of administration sufficient to effectivelystimulate a cytotoxic T cell response; compositions which stimulatehelper T cell responses can also be given in accordance with thisembodiment of the invention.

[0147] The dosage for an initial therapeutic immunization generallyoccurs in a unit dosage range where the lower value is about 1, 5, 50,500, or 1000 μg of peptide and the higher value is about 10,000; 20,000;30,000; or 50,000 μg. Dosage values for a human typically range fromabout 500 μg to about 50,000 μg per 70 kilogram patient. Boostingdosages of between about 1.0 μg to about 50000 μg of peptide pursuant toa boosting regimen over weeks to months may be administered dependingupon the patient's response and condition as determined by measuring thespecific activity of CTL and HTL obtained from the patient's blood. Thepeptides and compositions of the present invention may be employed inserious disease states, that is, life-threatening or potentially lifethreatening situations. In such cases, as a result of the minimalamounts of extraneous substances and the relative nontoxic nature of thepeptides in preferred compositions of the invention, it is possible andmay be felt desirable by the treating physician to administersubstantial excesses of these peptide compositions relative to thesestated dosage amounts.

[0148] The pharmaceutical compositions for therapeutic treatment areintended for parenteral, topical, oral, intrathecal or localadministration. Preferably, the pharmaceutical compositions areadministered parentally, e.g. intravenously, subcutaneously,intradermally, or intramuscularly. Thus, the invention providescompositions for parenteral administration which comprise a solution ofthe immunogenic peptides dissolved or suspended in an acceptablecarrier, preferably an aqueous carrier. A variety of aqueous carriersmay be used, e.g. water, buffered water, 0.8% saline, 0.3% glycine,hyaluronic acid and the like. These compositions may be sterilized byconventional, well-known sterilization techniques, or may be sterilefiltered. The resulting aqueous solutions may be packaged for use as is,or lyophilized, the lyophilized preparation being combined with asterile solution prior to administration. The compositions may containpharmaceutically acceptable auxiliary substances as required toapproximate physiological conditions, such as pH-adjusting and bufferingagents, tonicity adjusting agents, wetting agents, preservatives, andthe like, for example, sodium acetate, sodium lactate, sodium chloride,potassium chloride, calcium chloride, sorbitan monolaurate,triethanolamine oleate, etc.

[0149] The concentration of peptides of the invention in thepharmaceutical formulations can vary widely, i.e., from less than about0.1%, usually at or at least about 2% to as much as 20% to 50% or moreby weight, and will be selected primarily by fluid volumes, viscosities,etc., in accordance with the particular mode of administration selected.

[0150] A human unit dose form of the peptide composition is typicallyincluded in a pharmaceutical composition that comprises a human unitdose of an acceptable carrier, preferably an aqueous carrier, and isadministered in a volume of fluid that is known by those of skill in theart to be used for administration of such compositions to humans (see,e.g., Remington's Pharmaceutical Sciences, 17^(th) Edition, A. Gennaro,Editor, Mack Publising Co., Easton, Pa., 1985).

[0151] The peptides of the invention may also be administered vialiposomes, which serve to target the peptides to a particular tissue,such as lymphoid tissue, or to target selectively to infected cells, aswell as to increase the half-life of the peptide composition. Liposomesinclude emulsions, foams, micelles, insoluble monolayers, liquidcrystals, phospholipid dispersions, lamellar layers and the like. Inthese preparations, the peptide to be delivered is incorporated as partof a liposome, alone or in conjunction with a molecule which binds to areceptor prevalent among lymphoid cells, such as monoclonal antibodieswhich bind to the CD45 antigen, or with other therapeutic or immunogeniccompositions. Thus, liposomes either filled or decorated with a desiredpeptide of the invention can be directed to the site of lymphoid cells,where the liposomes then deliver the peptide compositions. Liposomes foruse in accordance with the invention are formed from standardvesicle-forming lipids, which generally include neutral and negativelycharged phospholipids and a sterol, such as cholesterol. The selectionof lipids is generally guided by consideration of, e.g., liposome size,acid lability and stability of the liposomes in the blood stream. Avariety of methods are available for preparing liposomes, as describedin, e.g. Szoka, et al., Ann. Rev. Biophys. Bioeng. 9:467 (1980), andU.S. Pat. Nos. 4,235,871, 4,501,728, 4,837,028, and 5,019,369.

[0152] For targeting cells of the immune system, a ligand to beincorporated into the liposome can include, e.g., antibodies orfragments thereof specific for cell surface determinants of the desiredimmune system cells. A liposome suspension containing a peptide may beadministered intravenously, locally, topically, etc. in a dose whichvaries according to, inter alia, the manner of administration, thepeptide being delivered, and the stage of the disease being treated.

[0153] For solid compositions, conventional nontoxic solid carriers maybe used which include, for example, pharmaceutical grades of mannitol,lactose, starch, magnesium stearate, sodium saccharin, talcum,cellulose, glucose, sucrose, magnesium carbonate, and the like. For oraladministration, a pharmaceutically acceptable nontoxic composition isformed by incorporating any of the normally employed excipients, such asthose carriers previously listed, and generally 10-95% of activeingredient, that is, one or more peptides of the invention, and morepreferably at a concentration of 25%-75%.

[0154] For aerosol administration, the immunogenic peptides arepreferably supplied in finely divided form along with a surfactant andpropellant Typical percentages of peptides are 0.01%-20% by weight,preferably 1%-10%. The surfactant must, of course, be nontoxic, andpreferably soluble in the propellant. Representative of such agents arethe esters or partial esters of fatty acids containing from 6 to 22carbon atoms, such as caproic, octanoic, lauric, palmitic, stearic,linoleic, linolenic, olesteric and oleic acids with an aliphaticpolyhydric alcohol or its cyclic anhydride. Mixed esters, such as mixedor natural glycerides may be employed. The surfactant may constitute0.1%-20% by weight of the composition, preferably 0.25-5%. The balanceof the composition is ordinarily propellant. A carrier can also beincluded, as desired, as with, e.g., lecithin for intranasal delivery.

EXAMPLES Example 1 Immunogenic Peptide Isolated from HPV-18E6 Protein

[0155] The peptide KLPDLCTEL(SEQ ID NO:1) identified in the HPV-18E6protein (amino acids 13-21) contains a predicted HLA-A2 binding motif.To determine if this peptide could bind to this class I MHC molecule,the peptide was synthesized, fluorescently labeled and tested forbinding to T2A2 cells. T2A2 cells are T cells which possess the HLA-A2molecule on their cell surface. Also tested in this binding assay werethe flu matrix peptide (FMP) which is known to bind to the HLA-A2molecule with high affinity and the peptide KLPQLCTEL (SEQ ID NO.10),from the HPV-16E6 protein (amino acids 13-21). The results are shown inFIG. 1. All three peptides bound to the HLA-A2 molecules on the surfaceof the T2A2 cells. Surprisingly, the peptide of the invention,KLPDLCTEL(SEQ ID NO:1), bound to the HLA-A2 molecule with a higheraffinity than did either the FMP peptide or the homologous peptide fromthe HPV-16E6 protein. The only difference between the HPV-18E6 andHPV-16E6 peptides is at amino acid position 4. The HPV-18E6 peptidecontains a D residue at amino acid 4 while the HPV-16E6 peptide containsa Q residue at this position. Because the KLPDLCTEL(SEQ ID NO:1) peptideisolated from HPV-18E6 bound to the HLA-A2 molecule with approximately100 fold higher affinity than did the homologous KLPQLCTEL (SEQ IDNO.10) peptide isolated from HPV-16E6, the KLPDLCTEL(SEQ ID NO:1)peptide should be more efficient at generating an immune response to HPVthan the homologous HPV-16E6 peptide.

[0156] To determine if lymphocytes stimulated in vitro with either theHPV-18E6 peptide (SEQ ID NO.1) or the HPV-16E6 peptide (SEQ ID NO. 10)would recognize and lyse peripheral blood mononuclear cells (PBMCs)pulsed with these peptides, the following experiment was performed.Lymphocytes isolated from a patient known to carry and express theHLA-A2 allele were stimulated in vitro (IVS) with either the HPV-18E6peptide (SEQ ID NO. 1) or the HPV-16E6 peptide (SEQ ID NO. 10) at a 10μm concentration for 1-3 weeks. Autologous PBMC target cells wereradiolabeled with ⁵¹Cr and pulsed with either the HPV-18E6 peptide (SEQID NO.1) or the HPV-16E6 peptide (SEQ ID NO. 10). Each of the IVSlymphocyte populations were then mixed with each of the pulsed andlabeled PBMC target cells and specific lysis was measured. The resultsare shown in FIG. 2. Lymphocytes IVS with either the HPV-18E6 peptide(SEQ ID NO. 1) or the HPV-16E6 peptide (SEQ ID NO.10) were able to lysePBMCs which were pulsed with either the HPV-18E6 peptide (SEQ ID NO. 1)or the HPV-16E6 peptide (SEQ ID NO. 10) and had either the HPV-18E6peptide (SEQ ID NO. 1) or the HPV-16E6 (SEQ ID NO. 10) peptide on theircell surface. These data indicate that the HPV-18E6 peptide (SEQ IDNO. 1) would be useful for evaluating T cell responses in patientsinfected with HPV, or individuals who have been vaccinated with eitherthe HPV-18E6 peptide (SEQ ID NO. 1) or the HPV-16E6 peptide (SEQ ID NO.10).

[0157] These data indicate that the HPV-18E6 peptide (SEQ ID NO. 1) isable to bind to HLA-A2 molecule on the cell surface and is able toelicit an immune response. This peptide, either alone, or in combinationwith other peptides (see Table 1) would be useful in vaccinecompositions and methods of using the peptides of Table 1. TABLE 1Immunogenic peptides. Peptide Class type SEQ ID NO. KLPDLCTEL Class ISEQ ID NO.1 (SEQ ID NO:1) DRAHYNI Class II SEQ ID NO.2 IVCPICSQ Class ISEQ ID NO.4 LLMGTLGIV Class I SEQ ID NO.5 TLGIVCPIC Class I SEQ ID NO.6LLMGTLGIVCP Class I SEQ ID NO.7 TLGIVCPI Class I SEQ ID NO.8 GTLGIVCPIClass I SEQ ID NO.9

[0158] Additional immunogenic peptides have been identified whichcontain motifs which bind to either class I or class II MHC molecules(see e.g., Kast, et al., J. Immunol. 152:3904-3912 (1994), U.S. Pat. No.6,183,746, and U.S. Pat. No. 6,004,557) and are found in Table I. Thesepeptides can be combined with the immunogenic peptide KLPDLCTEL(SEQ IDNO:1) isolated from HPV-18E6 protein to create polyepitopic peptideswhich would bind to multiple HLA class I molecules or to both HLA classI (CTL) and class II (HTL) molecules. The peptides can be directlylinked to the peptide KLPDLCTEL(SEQ ID NO:1) or be connected via aspacer peptide. Spacers are typically selected from e.g., Ala, Gly orother neutral spacers of non-polar amino acids or neutral polar aminoacids. It is understood that the optionally present spacer need not becomprised of the same residues and thus may be a hetero- orhomo-oligomer. When present the spacer will usually be at least one totwo residues, and more usually three to six residues. The class I (CTL)peptide epitope can be linked to the class II (HTL) peptide epitopeeither directly or via a spacer at the amino or carboxy terminus of theclass I peptide.

[0159] As discussed above, the immunogenic peptides of Table I can becombined to create a polyepitopic peptide of 20 amino acids or less andof the structure X₁KLPDLCTEL(SEQ ID NO:1)X₂ where X₁ and X₂ are peptidesof 0-11 amino acid residues in length and can include a spacer sequence.

Example 2 Peptide Composition for Prophylactic Uses

[0160] Vaccine compositions of the present invention are used to preventHPV infection in persons who are at risk for such infection. Forexample, a monoepitopic peptide or a polyepitopic peptide epitopecomposition (or a nucleic acid comprising the same) containing a singleHPV-18E6 epitope or multiple CTL and/or HTL epitopes such as those inTable 1, is administered to individuals at risk for HPV infection. Thecomposition is provided as a single lipidated polypeptide thatencompasses the single or multiple epitopes. The vaccine is administeredin an aqueous carrier comprised of Freunds Incomplete Adjuvant The doseof peptide for the initial immunization is from about 1 to about 50,000μg, generally 100-5,000 μg, for a 70 kg patient. The initialadministration of vaccine is followed by booster dosages at 4 weeksfollowed by evaluation of the magnitude of the immune response in thepatient, by techniques that determine the presence of epitope-specificCTL populations in a PBMC sample. Additional booster doses areadministered as required. The composition is found to be both safe andefficacious as a prophylaxis against HPV infection.

[0161] Alternatively, the monoepitopic peptide or the polyepitopicpeptide compositions can be administered as a nucleic acid in accordancewith methodologies known in the art and disclosed herein.

Example 3 Construction of Minizene Multi-Epitope DNA Plasmids

[0162] This example provides guidance for the construction of a minigeneexpression plasmid. Minigene plasmids may, of course, contain variousconfigurations of CTL and/or HTL epitopes or epitope analogs asdescribed herein.

[0163] A minigene expression plasmid may include multiple CTL and/or HTLpeptide epitopes. Preferred epitopes are identified, for example, inTable 1. The selected CTL and HTL epitopes are then incorporated into aminigene for expression in an expression vector.

[0164] This example illustrates the methods to be used for constructionof such a minigene-bearing expression plasmid. Other expression vectorsthat may be used for minigene compositions are available and known tothose of skill in the art.

[0165] The minigene DNA plasmid contains a consensus Kozak sequence anda consensus murine kappa Ig-light chain signal sequence followed by CTLand/or HTL epitopes selected in accordance with principles disclosedherein.

[0166] Overlapping oligonucleotides encoding the selected peptides aresynthesized and HPLC-purified. The oligonucleotides encode the selectedpeptide epitopes as well as appropriate linker nucleotides, Kozaksequence, signal sequence, and stop codon. The final multiepitopeminigene is assembled by extending the overlapping oligonucleotides inreactions using PCR. A Perkin/Elmer 9600 PCR machine is used and a totalof 30 cycles are performed using the following conditions: 95° C. for 15sec, annealing temperature (5° below the lowest calculated Tm of eachprimer pair) for 30 sec, and 72° C. for 1 min.

[0167] For the PCR reaction, 5 μg of each of the two oligonucleotidesare annealed and extended: Oligonucleotides are combined in 100 μlreactions containing Pfu polymerase buffer (1x=10 mM KCL, 10 mM(NH₄)₂SO₄, 20 mM Tris-chloride, pH 8.75, 2 mM MgSO₄, 0.1% Triton X-100,100 μg/ml BSA), 0.25 mM each dNTP, and 2.5 U of Pfu polymerase. Thefull-length dimer product is gel-purified and cloned into pCR-blunt(Invitrogen) and individual clones are screened by sequencing.

Example 4 The Plasmid Construct and the Degree to Which it InducesImmunogenicity

[0168] The degree to which the plasmid construct prepared using themethodology outlined in Example 3 is able to induce immunogenicity isevaluated through in vivo injections into mice and subsequent in vitroassessment of CTL and HTL activity, which are analyzed usingcytotoxicity and proliferation assays, respectively, as detailed e.g.,Alexander et al., Immunity 1:751-761, 1994. For example, to assess thecapacity of a pMin minigene construct that contains the HLA-A2 motifepitope KLPDLCTEL(SEQ ID NO:1) to induce CTLs in vivo, HLA-A2.1/Kbtransgenic mice are immunized intramuscularly with 100 μg of naked cDNA.As a means of comparing the level of CTLs induced by cDNA immunization,a control group of animals is also immunized with an actual peptidecomposition that comprises the epitope synthesized as a singlepolypeptide as they would be encoded by the minigene.

[0169] Splenocytes from immunized animals are stimulated twice with eachof the respective compositions (peptide epitopes encoded in the minigeneor the peptide), then assayed for peptide-specific cytotoxic activity ina ⁵¹Cr release assay. The results indicate the magnitude of the CTLresponse directed against the A2-restricted epitope, thus indicating thein vivo immunogenicity of the minigene vaccine and polypeptide vaccine.It is, therefore, found that the minigene elicits immune responsesdirected toward the HLA-A2 peptide epitope as does the peptide vaccine.

[0170] To assess the capacity of a class II epitope encoding minigene toinduce HTLs in vivo, I-A^(b) restricted mice, for example, are immunizedintramuscularly with 100 μg of plasmid DNA. As a means of comparing thelevel of HTLs induced by DNA immunization, a group of control animals isalso immunized with an actual peptide composition emulsified in completeFreund's adjuvant.

[0171] CD4+ T cells, i.e. HTLs, are purified from splenocytes ofimmunized animals and stimulated with each of the respectivecompositions (peptides encoded in the minigene). The HTL response ismeasured using a ³H-thymidine incorporation proliferation assay, (see,e.g., Alexander et al. Immunity 1:751-761, 1994) the results indicatethe magnitude of the HTL response, thus demonstrating the in vivoimmunogenicity of the minigene.

[0172] Alternatively, plasmid constructs can be evaluated in vitro bytesting for epitope presentation by APC following transduction ortransfection of the APC with an epitope-expressing nucleic acidconstruct. Such a study determines “antigenicity” and allows the use ofhuman APC. The assay determines the ability of the epitope to bepresented by the APC in a context that is recognized by a T cell byquantifying the density of epitope-HLA class I complexes on the cellsurface. Quantitation can be performed by directly measuring the amountof peptide eluted from the APC (see, e.g., Sijts et al., J. Immunol.156:683-692, 1996; Demotz et al., Nature 342:682-684, 1989); or thenumber of peptide-HLA class I complexes can be estimated by measuringthe amount of lysis or lymphokine release induced by infected ortransfected target cells, and then determining the concentration ofpeptide necessary to obtained equivalent levels of lysis or lymphokinerelease (see, e.g., Kageyama et al., J. Immunol. 154:567-576, 1995).

Example 5 Use of Peptides to Evaluate an Immune Response

[0173] In-vitro T Cell Cytotoxic Assay:

[0174] T cell cytotoxicity is measured by the standard ⁵¹Cr-releaseassay. Briefly, target cells are radiolabeled with Na₂ ⁵¹CrO₄. Thepatient's PBMC (which have been previously stimulated with an HPVpeptide of Example 1 at 10 μm concentration for 1 to 3 weeks) are addedto the labeled target cells in the presence or absence of the peptide.Cell lysis is determined by the specific release of ⁵¹Cr (specificlysis). Target cells are autologous B-LCL or T2 cells pulsed with thepeptide. If there is a detectable pre-immunization specific lysis, a 2.0fold increase in the lytic units will be considered a positive response.If there is no detectable pre-immunization specific lysis, apost-immunization specific lysis of less than 10% above the controlswill be considered a positive response.

[0175] Assessment of IFN-Gamma Response:

[0176] To assess the IFN-gamma response, 3×10⁶ PBMC per well arecultured in a 24-well plate±HPV peptide of Example 1/or tetanus toxoid+20 IU/ml IL-2 for 6-19 days period. For positive control wells, PBMCare cultured in wells containing FMP with IL-2 and without HPV peptideof Example 1. Culture supernatants are harvested and stored frozen at(−20° C.) until assayed for IFN-gamma by ELISA. Titrated amounts ofculture supernatants are tested using commercially available ELISA kits(Endogen, GIBCO, or Genzyme). The amount of IFN-gamma in culturesupernatants is determined by comparing experimental results to standardcurves generated with known amounts of recombinant human IFN-gamma.Stimulator cells will be both autologous PBMC pulsed with peptides andautologous tumor, if available. A positive response will be taken as a2-fold increase over background as long as at least 50 pg/ml are made.The intra-assay variability is less than 5%.

[0177] In-Vitro T Cell Proliferation Assay:

[0178] An autologous patients PBMC is incubated in-vitro with tetanustoxoid (Tt) and evaluated for Tt-induced proliferation following up to 5days of incubation. Cultures are pulsed with ³H-thymidine for the final18-24 hours of their culture. Proliferation is measured and quantitatedby the incorporation of ³H-thymidine. A proliferation of more than threefold above control will be considered as a positive response.

Example 6 Use of Peptide Epitopes to Evaluate Recall Responses

[0179] The peptide epitopes of Example 1 are used as reagents toevaluate T cell responses, such as acute or recall responses, inpatients. Such an analysis may be performed on patients who haverecovered from infection, who have been vaccinated with an HPV vaccineof the invention, or who have been vaccinated containing peptideepitopes homologous to the peptides in Table 1.

[0180] PBMC from vaccinated individuals or individuals who haverecovered from an infection are separated on Ficoll-Histopaque densitygradients (Sigma Chemical Co., St. Louis, Mo.), washed three times inHBSS (GIBCO Laboratories), resuspended in RPMI-1640 (GIBCO Laboratories)supplemented with L-glutamine (2 mM), penicillin (50U/ml), streptomycin(50 μg/ml), and Hepes (10 mM) containing 10% heat-inactivated human ABserum (complete RPMI) and plated using microculture formats. A syntheticpeptide comprising an epitope of the invention is added at 10 μg/ml toeach well and DRAHYNI (SEQ ID NO. 2) is added at 1 μg/ml to each well asa source of T cell help during the first week of stimulation.

[0181] In the microculture format, 4×10⁵ PBMC are stimulated withpeptide in 8 replicate cultures in 96-well round bottom plate in 100μl/well of complete RPMI. On days 3 and 10, 100 ml of complete RPMI and20 U/ml final concentration of rIL-2 are added to each well. On day 7the cultures are transferred into a 96-well flat-bottom plate andrestimulated with peptide, rIL-2 and 10⁵ irradiated (3,000 rad)autologous feeder cells. The cultures are tested for cytotoxic activityon day 14. A positive CTL response requires two or more of the eightreplicate cultures to display greater than 10% specific ⁵¹Cr release,based on comparison with uninfected control subjects as previouslydescribed (Rehermann, et al., Nature Med. 2:1104,1108, 1996; Rehermannet al., J. Clin. Invest. 97:1655-1665, 1996; and Rehermann et al J.Clin. Invest. 98:1432-1440, 1996).

[0182] Target cell lines are autologous and allogeneic EBV-transformedB-LCL that are either purchased from the American Society forHistocompatibility and Immunogenetics (ASHI, Boston, Mass.) orestablished from a pool of patients as described (Guilhot, et al. J.Virol 66:2670-2678, 1992) or T2 cells.

[0183] Cytotoxicity assays are performed in the following manner. Targetcells consist of either allogeneic HLA-matched or autologousEBV-transformed B lymphoblastoid cell line that are incubated overnightwith the synthetic peptide epitope of the invention at 10 μM, andlabeled with 100 μCi of ⁵¹Cr (Amersham Corp., Arlington Heights, Ill.)for 1 hour after which they are washed four times with HBSS.

[0184] Cytolytic activity is determined in a standard 4-h, split well⁵¹Cr release assay using U-bottomed 96 well plates containing 3,000targets/well. Stimulated PBMC are tested at effector/target (E/T) ratiosof 20-50:1 on day 14. Percent cytotoxicity is determined from theformula: 100×[(experimental release-spontaneous release)/maximumrelease-spontaneous release)]. Maximum release is determined by lysis oftargets by detergent (2% Triton X-100; Sigma Chemical Co., St. Louis,Mo.). Spontaneous release is <25% of maximum release for allexperiments.

[0185] The results of such an analysis indicate the extent to whichHLA-restricted CTL populations have been stimulated by previous exposureto HPV or an HPV vaccine.

[0186] The class II restricted HTL responses may also be analyzed.Purified PBMC are cultured in a 96-well flat bottom plate at a densityof 1.5×10⁵ cells/well and are stimulated with 10 μg/ml synthetic peptidecontaining the DRAHYNI (SEQ ID NO.2) peptide, or PHA. Cells areroutinely plated in replicates of 4-6 wells for each condition. Afterseven days of culture, the medium is removed and replaced with freshmedium containing 10U/ml IL-2. Two days later, 1 μCi³H-thymidine isadded to each well and incubation is continued for an additional 18hours. Cellular DNA is then harvested on glass fiber mats and analyzedfor ³H-thymidine incorporation. Antigen-specific T cell proliferation iscalculated as the ratio of ³H-thymidine incorporation in the presence ofantigen divided by the ³H-thymidine incorporation in the absence ofantigen.

Example 7 Induction Of Specific CTL Response In Humans

[0187] A human clinical trial for an immunogenic composition comprisingCTL and HTL epitopes of Example 1, including SEQ ID NO:1, is set up asan IND Phase I, dose escalation study and carried out as a randomized,double-blind, placebo-controlled trial. Such a trial is designed, forexample, as follows:

[0188] A total of about 27 subjects are enrolled and divided into 3groups:

[0189] Group I: 3 subjects are injected with placebo and 6 subjects areinjected with 5 μg of peptide composition;

[0190] Group II: 3 subjects are injected with placebo and 6 subjects areinjected with 50 μg peptide composition;

[0191] Group m: 3 subjects are injected with placebo and 6 subjects areinjected with 500 μg of peptide composition.

[0192] After 4 weeks following the first injection, all subjects receivea booster inoculation at the same dosage.

[0193] The endpoints measured in this study relate to the safety andtolerability of the peptide composition as well as its immunogenicity.Cellular immune responses to the peptide composition are an index of theintrinsic activity of this the peptide composition, and can therefore beviewed as a measure of biological efficacy. The following summarize theclinical and laboratory data that relate to safety and efficacyendpoints.

[0194] Safety: The incidence of adverse events is monitored in theplacebo and drug treatment group and assessed in terms of degree andreversibility.

[0195] Evaluation of Vaccine Efficacy: For evaluation of vaccineefficacy, subjects are bled before and after injection. Peripheral bloodmononuclear cells are isolated from fresh heparinized blood byFicoll-Hypaque density gradient centrifugation, aliquoted in freezingmedia and stored frozen. Samples are assayed for CTL and HTL activity.

[0196] The vaccine is found to be both safe and efficacious.

Example 8 Administration of Vaccine Compositions Using Dendritic Cells

[0197] Vaccines comprising peptide epitopes of Example 1, including SEQID NO:1, may be administered using dendritic cells. In this example, thepeptide-pulsed dendritic cells can be administered to a patient tostimulate a CTL response in vivo. In this method dendritic cells areisolated, expanded, and pulsed with a vaccine comprising peptide CTL andHTL epitopes of the invention. The dendritic cells are infused back intothe patient to elicit CTL and HTL responses in vivo. The induced CTL andHTL then destroy (CTL) or facilitate destruction (HTL) of the specifictarget HPV-infected cells that bear the proteins from which the epitopesin the vaccine are derived.

[0198] Alternatively, Ex vivo CTL or HTL responses to a particulartumor-associated antigen can be induced by incubating in tissue culturethe patients, or genetically compatible, CTL or HTL precursor cellstogether with a source of antigen-presenting cells, such as dendriticcells, and the appropriate immunogenic peptides. After an appropriateincubation time (typically about 7-28 days), in which the precursorcells are activated and expanded into effector cells, the cells areinfused back into the patient, where they will destroy (CTL) orfacilitate destruction (HTL) of their specific target cells, i.e., tumorcells.

[0199] It is understood that the examples and embodiments describedherein are for illustrative purposes only and that various modificationsor changes in light thereof will be suggested to persons skilled in theart and are to be included within the spirit and purview of thisapplication and scope of the appended claims. All publications, patentsand patent applications cited herein are hereby incorporated byreference.

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What is claimed is:
 1. A method of inducing a cytotoxic T lymphocyteresponse against human papilloma virus 16 (HPV 16) in a patient, themethod comprising contacting cytotoxic T cells from a patient with animmunogenic peptide of 20 amino acid residues or less comprising across-reactive peptide from the E6 protein of a related HPV strain, HPV18, that has higher affinity for the HLA-A2.1 molecule than thecorresponding epitope from HPV 16 itself, said peptide comprising thesequence X₁KLPDLCTEL(SEQ ID NO:1)X₂, wherein X₁ and X₂ are peptides of0-11 amino acids in length comprising either native or non-native aminoacid sequence and returning said cytotoxic T cells to the patient in anamount sufficient to induce a cytotoxic T cell response.
 2. The methodof claim 1, wherein X₁ or X₂ comprises an HLA binding motif other thanHLA-A2.
 3. The method of claim 1 wherein X₁ or X₂ comprises an aminoacid sequence capable of binding to an HLA class II molecule
 4. Themethod of claim 1, wherein said peptide is bound to an HLA molecule onan antigen presenting cell.
 5. The method of claim 1, wherein saidpeptide is bound to an HLA molecule on a lymphocyte.
 6. The method ofclaim 5, wherein said HLA molecule is HLA-A2.
 71. The method of claim 5,wherein said HLA molecule is an HLA molecule other than HLA-A2.
 8. Amethod for inducing an immune response against human papilloma virus 16(HPV 16) comprising administering to a subject a composition, which isselected from a group consisting of: (i) a peptide of 20 amino acids orless comprising a cross-reactive peptide from the E6 protein of arelated HPV strain, HPV 18, that has higher affinity for the HLA-A2.1molecule than the corresponding epitope from HPV 16 itself, said peptidecomprising the sequence X₁KLPDLCTEL(SEQ ID NO:1)X₂, wherein X₁ and X₂are peptides of 0-11 amino acids in length comprising either native ornon-native amino acid sequences; (ii) an antigen presenting cell pulsedwith said peptide; and (iii) a cell sensitized in vitro to said peptide.9. The method of claim 8, wherein the composition is in apharmaceutically acceptable carrier.
 10. The method of claim 8, whereinthe composition is in a sterile medium.
 11. The method of claim 9,further comprising co-administering to the subject an immune adjuvantselected from non-specific immune adjuvants, subcellular microbialproducts and fractions, haptens, immunogenic proteins, immunomodulators,interferons, thymic hormones and colony stimulating factors.
 12. Themethod of claim 8, wherein the administration comprises sensitizing CD8+cells in vitro to said peptide and administering the sensitized cells tothe subject in a sterile medium.
 13. A vaccine for preventing ortreating human papilloma virus 16 (HPV 16) infection that induces aprotective or therapeutic immune response, wherein said vaccinecomprises a peptide of 20 amino acids or less comprising across-reactive peptide from the E6 protein of a related HPV strain, HPV18, that has higher affinity for the HLA-A2.1 molecule than thecorresponding epitope from HPV 16 itself, said peptide comprising thesequence X₁KLPDLCTEL(SEQ ID NO:1)X₂, wherein X₁ and X₂ are peptides of0-11 amino acids in length comprising either native or non-native aminoacid sequences and a pharmaceutically acceptable carrier.
 14. Thevaccine of claim 13 further comprising co-administering to the subjectan immune adjuvant selected from non-specific immune adjuvants,subcellular microbial products and fractions, haptens, immunogenicproteins, immunomodulators, interferons, thymic hormones and colonystimulating factors.
 15. The vaccine of claim 14, wherein said peptideis administered by administering to a subject an expression vector thatexpresses said peptide.
 16. A method for monitoring or evaluating animmune response to human papilloma virus 16 (HPV 16) in a patient havingthe HLA-A2.1 type, the method comprising incubating a T lymphocytesample from the patient with a peptide of 20 amino acids or lesscomprising a cross-reactive peptide from the E6 protein of a related HPVstrain, HPV 18, that has higher affinity for the HLA-A2.1 molecule thanthe corresponding epitope from HPV 16 itself, said peptide comprisingthe sequence X₁KLPDLCTEL(SEQ ID NO:1)X₂, wherein X₁ and X₂ are peptidesof 0-11 amino acids in length comprising either native or non-nativeamino acid sequences and which peptide bears a binding motifcorresponding to at least one HLA allele present in said patient, anddetecting the presence of a T lymphocyte that recognizes the peptide.17. A method screening for exposure to human papilloma virus 16 (HPV 16)in a patient having the HLA-A2.1 type, the method comprising incubatinga T lymphocyte sample from the patient with a peptide of 20 amino acidsor less comprising a cross-reactive peptide from the E6 protein of arelated HPV strain, HPV 18, that has higher affinity for the HLA-A2.1molecule than the corresponding epitope from HPV 16 itself, said peptidecomprising the sequence X₁KLPDLCTEL(SEQ ID NO:1)X₂, wherein X₁ and X₂are peptides of 0-11 amino acids in length comprising either native ornon-native amino acid sequences and which peptide bears a binding motifcorresponding to at least one HLA allele present in said patient, anddetecting the presence of a T lymphocyte that recognizes the peptide,the presence of such a T lymphocyte indicating exposure to HPV
 18. Amethod of inducing a cytotoxic T lymphocyte response and protectiveimmunity against tumors induced by human papilloma virus 16 (HPV 16)using a cross-reactive peptide from the E6 protein of a related HPVstrain, HPV 18, that has higher affinity for the HLA-A2.1 molecule thanthe corresponding epitope from HPV 16 itself, said HPV18 peptide havingthe sequence X₁KLPDLCTEL(SEQ ID NO:1)X₂, wherein X₁ and X₂ are peptidesof 0-11 amino acids.